Screening of UBE2S interacting protein and construction of prognostic model in hepatocellular carcinoma / 吉林大学学报(医学版)

Objective:To screen the interacting protein of ubiquitin-conjugating enzyme E2S(UBE2S)and construct the hepatocellular carcinoma(HCC)based on UBE2S interacting protein prognosis model(UIPM),and to discuss the value of UIPM in assessing the prognosis of the HCC patients.Methods:Co-immunoprecipitation(Co-IP)was used to screen the protein complexes binding to Flag-UBE2S.After validation by sodium dodecyl sulphate-polyacrylamide gel electrophoresis(SDS-PAGE)and Western blotting methods;liquid chromatography-mass spectrometer(LC-MS)was used to identify the UBE2S interacting proteins;Gene Ontology(GO)functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)signaling pathway enrichment analysis were conducted on these proteins;the prognosis-related proteins from The Cancer Genome Atlas(TCGA)were cross-referenced with UBE2S interacting proteins by survival package of R software;the key proteins were extracted through LASSO regression analysis to build the UIPM;the prognostic model risk scoring formula was established.The HCC patients in TCGA were divided into high risk group and low risk group based on median value of the risk scores.The predictive accuracy of UIPM was evaluated by receiver operating characteristic curve(ROC),and the predictive accuracy was further validated by International Cancer Genome Consortium(ICGC)Database;univariate regression analysis and multivariate Cox regression analysis were used to detect whether the UIPM risk score was an independent prognostic factor for HCC.Furthermore,the nomogram model was built.Results:A total of 97 UBE2S interacting proteins were identified through Co-IP combined with LC-MS analysis.The GO functional enrichment analysis and KEGG signaling pathway enrichment analysis results showed that the interacting proteins were closely associated with cysteine-type endopeptidase activity,oxidative stress,and cell death.The TCGA revealed 5 163 HCC prognosis-related proteins;after intersecting with UBE2S interacting proteins,40 prognosis-related interacting proteins were found.Seven key proteins were determined through LASSO regression analysis,including UBE2S,heat shock protein family A member 8(HSPA8),heterogeneous nuclear ribonucleoprotein H1(HNRNPH1),chaperonin containing TCP1 subunit 3(CCT3),eukaryotic translation initiation factor 2 subunit 1(EIF2S1),receptor for activated C kinase 1(RACK1),and actin related protein 2/3 complex subunit 4(ARPC4),and the UIPM was constructed.There was significant difference in survival rate of the patients between high risk group and low risk group(P<0.05).The ROC curve analysis results showed the area under ROC curve(AUC)values of UIPM for predicting 1-year,2-year,and 3-year survival risk scores of the HCC patients were all greater than 0.7,indicating the model had high predictive accuracy.This was also confirmed by ICGC Database data.The univariate and multivariate Cox regression analysis results showed that the UIPM risk score was an independent prognostic risk factor for the HCC patients(P<0.05).The nomogram results showed good consistency between predicted survival rate and actual survival rate of the patient.Conclusion:A total of 97 interacting proteins that interact with UBE2S may promote the occurence and devolopment of HCC through oxidative stress and dysregulation of ferroptosis pathways.The UIPM risk score is an independent risk factor for the prognosis of HCC and can be used to predict the outcomes of the patients.UBE2S,HSPA8,HNRNPH1,CCT3,EIF2S1,RACK1,and ARPC4 could be regarded as the new biomarkers and therapeutic targets for HCC.

Expression pattern and signification of Cx43,beta-catenin and Smo in the second heart field / 中国组织工程研究

BACKGROUND:The second heart field is crucial for the development of the embryonic heart.Abnormal development of the second heart field can result in multiple cardiac malformations.After Cx43 gene knockout,reduced formation and proliferation of cells of the second heart field can be observed,but the specific reason remains unclear. OBJECTIVE:(1)To determine whether β-catenin,Smo and Cx43 were co-expressed in the second heart field and the endoderm,we observed the expression patterns of these proteins.(2)To explore whether Cx43 interacts with the Wnt/β-catenin pathway or the Shh pathway in the development of the second heart field. METHODS:Serial paraffin sections of the mouse embryos at embryonic days 10-12 were selected for immunohistochemical staining,hematoxylin-eosin staining and immunofluorescence staining.The primitive gut of mouse embryos at embryonic day 11 was separated for western blot assay and co-immunoprecipitation. RESULTS AND CONCLUSION:(1)Cx43 and Isl1 were co-expressed in some mesenchymal cells on the ventral side of the foregut and dorsal wall of the pericardial cavity of mouse embryos at embryonic days 10-12;Isl1 positive cells increased while Cx43 positive cells increased.(2)Cx43 and β-catenin were co-expressed in the ventral part of the endoderm at embryonic days 10-12.(3)Cx43 and Smo were co-expressed in the endoderm at embryonic days 10-12.(4)The co-immunoprecipitation results confirmed that there was an interaction between Cx43 and β-catenin,which suggested that Cx43 interacted with β-catenin to participate in the development of the second heart field.

Peroxidase from foxtail millet bran exerts anti-colorectal cancer activity via targeting cell-surface GRP78 to inactivate STAT3 pathway

Molecular targeted therapy has become an emerging promising strategy in cancer treatment, and screening the agents targeting at cancer cell specific targets is very desirable for cancer treatment. Our previous study firstly found that a secretory peroxidase of class III derived from foxtail millet bran (FMBP) exhibited excellent targeting anti-colorectal cancer (CRC) activity in vivo and in vitro, whereas its underlying target remains unclear. The highlight of present study focuses on the finding that cell surface glucose-regulated protein 78 (csGRP78) abnormally located on CRC is positively correlated with the anti-CRC effects of FMBP, indicating it serves as a potential target of FMBP against CRC. Further, we demonstrated that the combination of FMBP with the nucleotide binding domain (NBD) of csGRP78 interfered with the downstream activation of signal transducer and activator of transcription 3 (STAT3) in CRC cells, thus promoting the intracellular accumulation of reactive oxygen species (ROS) and cell grown inhibition. These phenomena were further confirmed in nude mice tumor model. Collectively, our study highlights csGRP78 acts as an underlying target of FMBP against CRC, uncovering the clinical potential of FMBP as a targeted agent for CRC in the future.

Pharmacologically targeting molecular motor promotes mitochondrial fission for anti-cancer

Mitochondrial shape rapidly changes by dynamic balance of fusion and fission to adjust to constantly changing energy demands of cancer cells. Mitochondrial dynamics balance is exactly regulated by molecular motor consisted of myosin and actin cytoskeleton proteins. Thus, targeting myosin-actin molecular motor is considered as a promising strategy for anti-cancer. In this study, we performed a proof-of-concept study with a natural-derived small-molecule J13 to test the feasibility of anti-cancer therapeutics

Relationship between DEAD-box helicase 5, transcription factor 12 and amyotrophic lateral sclerosis / 解剖学报

Objective To explore the relationship between the expression of DEAO-box helicase 5(DDX5) and transcription factor 12(TCF12) with amyotrophic lateral sclerosis ( ALS ) hippocampal lesions by detecting the expressions and the interaction of DDX5 and TCF12 in the hippocampus of SOD1-G93A mutant ALS transgenic mice. Methods Forty- two pairs of SOD1-G93A mutant ALS transgenic mice and wild-type mice were divided into three groups at the age of 95 days (early onset stage), 108 days (middle onset stage) and 122 days (late onset stage). RT-PCR, Western blotting and double immunofluorescence labeled technique were used to detect the expressions of DDX5 and TCF12 in the hippocampus. Co-immunoprecipitation assasy was used to detect the interaction between DDX5 and TCF12. Results Compared with the wild-type mice of the same age, DDX5 and TCF12 mRNA in the hippocampus of SOD1-G93A mutant ALS transgenic mice were unchanged, but DDX5 and TCF12 protein were up-regulated significantly at day 95, 108 and 122. DDX5 and TCF12 positive cells were found in both DG area and hippocampus proper, and DDX5 and TCF12 were co-localized with neurons. The immunoreactivities of DDX5 and TCF12 in the hippocampus of SOD1-G93A mutant transgenic mice were elevated compared with wild-type mice at the same time point. Co-immunoprecipitation assasys confirmed that there existed interactions between DDX5 and TCF12 protein. Conclusion DDX5 and TCF12 protein are up-regulated in the hippocampal tissues of SOD1-G93A mutant ALS transgenic mice. The abnormal expressions of DDX5 and TCF12 are involved in the hippocampal lesions of ALS.

Eukaryotic expression, Co-IP and MS identify BMPR-1B protein-protein interaction network

BACKGROUND: BMPR-1B is part of the transforming growth factor ß super family and plays a pivotal role in ewe litter size. Functional loss of exon-8 mutations in the BMPR-1B gene (namely the FecB gene) can increase both the ewe ovulation rate and litter size. RESULTS: This study constructed a eukaryotic expression system, prepared a monoclonal antibody, and characterized BMPR-1B/FecB protein-protein interactions (PPIs). Using Co-immunoprecipitation coupled to mass spectrometry (Co-IP/MS), 23 proteins were identified that specifically interact with FecB in ovary extracts of ewes. Bioinformatics analysis of selected PPIs demonstrated that FecB associated with several other BMPs, primarily via signal transduction in the ovary. FecB and its associated interaction proteins enriched the reproduction process via BMP2 and BMP4 pathways. Signal transduction was identified via Smads proteins and TGF-beta signaling pathway by analyzing the biological processes and pathways. Moreover, other target proteins (GDF5, GDF9, RhoD, and HSP 10) that interact with FecB and that are related to ovulation and litter size in ewes were identified. CONCLUSIONS: In summary, this research identified a novel pathway and insight to explore the PPi network of BMPR-1B.

Aurone derivatives as Vps34 inhibitors that modulate autophagy

We report in this study the identification of a natural product-like antagonist () of Vps34 as a potent autophagy modulator structure-based virtual screening. Aurone derivative strongly inhibited Vps34 activity in cell-free and cell-based assays. Significantly, prevents autophagy in human cells induced either by starvation or by an mTOR inhibitor. modeling and kinetic data revealed that could function as an ATP-competitive inhibitor of Vps34. Moreover, it suppressed autophagy and without inducing heart or liver damage in mice. could be utilized as a new motif for more selective and efficacious antagonists of Vps34 for the potential treatment of autophagy-related human diseases.

Screening of S-phase kinase-assuciated protein 2 in the cervical carcinoma HeLa cell / 中国肿瘤生物治疗杂志

@#[Abstract] Objective： ：The co-immunoprecipitation and mass spectrometric analysis was carried out to obtain the S-phase kinase-associated protein 2 (SKP2)-binding proteins in HeLa cells, and the biological functions of these binding proteins were forecast. Methods: The co-immunoprecipitation system was established by co-immunoprecipitation and Western blotting assay; the specific protein gel of SKP2-binding proteins was obtained by SDS-PAGE and silver staining assay; the potential SKP2-binding proteins was identified by mass spectrometric analysis; and the GO (Gene ontology) analysis and KEGG analysis was carried out by bioinformatics technique. Results: The expression level of SKP2 protein in HeLa cells was high enough for co-immunoprecipitation assay; the co-immunoprecipitation system was established successfully, and SKP2-binding proteins was obtained; a total of 563 proteins were identified by mass spectrometric analysis, and 270 proteins with high credibility were obtained after screening. The GO analysis and KEGG analysis was carried out for the 270 proteins to forecast their functions and pathways. Conclusion: The SKP2-binding proteins were screened successfully, and it was the foundation for the subsequent screening of target-binding proteins and the search for targeting drugs.

Screening and verifying the proteins interacting with phosphorylation cluster of DNA-PKcs by yeast two-hybrid assay / 中华放射医学与防护杂志

Objective To screen and verify the proteins interacting with phosphorylation cluster of DNA dependent protein kinase catalytic subunit((DNA-PKcs) by yeast two-hybrid assay.Methods To know the proteins interacting with DNA-PKcs phosphorylation cluster,yeast two-hybrid assay was applied to screen the cDNA library of human hepatic tissue with a previously constructed plasmid pGBKT7-DPC.The positive clones were further identified by PCR,rotary validation and sequence analysis.Then the eukaryotic expression vectors of the bait protein and screened positive clone proteins were constructed and transfected into human embryonic kidney 293T cells to detect whether the proteins could been expressed correctly.At last,the bait protein and screened positive clone proteins were co-transfected into 293T cells and protein interaction was detected with Co-Immunoprecipitation (Co-IP) assay.Results After two rounds of screening using the yeast two-hybrid assay,12 candidate clones were obtained.Then 7 clones with different insert fragments were identified by PCR,and 3 positive proteins interacted with DNA-PKcs phosphorylation cluster were further verified by rotary validation.Sequencing analysis demonstrated that these 3 proteins were MBNL1,SIK2 and YY1AP1,respectively.Accordingly,the eukaryotic expression vectors of bait protein and 3 positive clone proteins were constructed successfully and expressed correctly in 293T ceils.Finally,the Co-IP assay confirmed that these 3 positive clone proteins could interact with DNA-PKcs phosphorylation cluster.Conclusions Proteins interacting with DNA-PKcs phosphorylation cluster are successfully screened and identified.

Effect of AR-V7 on endocrine therapy resistance of prostate cancer / 中国病理生理杂志

AIM:To explore the effect of androgen receptor splice variant 7 ( AR-V7 ) on endocrine therapy resistance of prostate cancer cells and the resistance mechanisms .METHODS:Four prostate cancer cell lines were trans-fected with AR-V7 siRNA ( siAR-V7) using Lipofectamine 2000 kit, and the transfected cells were named as PC 3-siAR-V7, DU145-siAR-V7, LNCaP-siAR-V7 and ArCaP-siAR-V7 cells.The prostate cancer cells transfected with negative con-trol ( NC) siRNA served as negative controls .The expression of AR-V7 at mRNA and protein levels was detected by real-time PCR and Western blot , respectively .The cell viability and cell migration rate were measured by MTT assay and Tran-swell method, respectively.The promoter activity of AR and the protein levels of targeted prostate-specific antigen (PSA) and FK506-binding protein 5 (FKBP5) were monitored by luciferase reporter gene assay and Western blot , respectively. The bicalutamide-resistant cell line, LNCaP-DR, was constructed, and the subcellular localization of AR and AR-V7 pro-teins in LNCaP, LNCaP-siAR-V7 and LNCaP-DR cells was observed by the method of immunofluorescence .The protein in-teraction of AR-V7 and heat shock protein 90 ( HSP90) was determined by co-immunoprecipitation .RESULTS:The mR-NA level of AR-V7 in the 4 prostate cancer cell lines was significantly higher than that in normal prostate epithelial cell line RWPE-1 (P<0.05).The AR-V7 level, cell viability and cell migration rate in the cells transfected with siAR-V7 were notablely lowered compared with the NC siRNA-transfected cells (P<0.05).The cell viability was gradually decreased following with the increase in bicalutamide dose , and down-regulation of AR-V7 expression significantly enhanced the sensi-tivity to bicalutamide (P<0.05).Down-regulation of AR-V7 expression significantly inhibited AR promoter activity and reduced the protein levels of PSA and FKBP5 (P<0.05).The results of immunofluorescence observation showed that most AR and AR-V7 were mainly located in the nucleus , a few AR was located in the cytoplasm , and down-regulation of AR-V7 expression inhibited AR nuclear transport .AR was entirely located in the nucleus and the protein levels of AR-V7 was sig-nificantly increased in the bicalutamide-resistant cells .The interaction of endogenous AR-V7 with HSP90 was found in the prostate cancer cells .CONCLUSION: High AR-V7 level is found in the prostate cancer cells , and down-regulation of AR-V7 expression inhibits the cell viability and migration .High AR-V7 level is related to the bicalutamide resistance .The possible mechanism is that AR nuclear transport is mediated by the interaction of AR -V7 with HSP90 to activate AR signal pathway and regulate targeted gene transcriptional activity , thus resulting in drug resistance .

Effect of AR-V7 on endocrine therapy resistance of prostate cancer / 中国病理生理杂志

AIM:To explore the effect of androgen receptor splice variant 7 ( AR-V7 ) on endocrine therapy resistance of prostate cancer cells and the resistance mechanisms .METHODS:Four prostate cancer cell lines were trans-fected with AR-V7 siRNA ( siAR-V7) using Lipofectamine 2000 kit, and the transfected cells were named as PC 3-siAR-V7, DU145-siAR-V7, LNCaP-siAR-V7 and ArCaP-siAR-V7 cells.The prostate cancer cells transfected with negative con-trol ( NC) siRNA served as negative controls .The expression of AR-V7 at mRNA and protein levels was detected by real-time PCR and Western blot , respectively .The cell viability and cell migration rate were measured by MTT assay and Tran-swell method, respectively.The promoter activity of AR and the protein levels of targeted prostate-specific antigen (PSA) and FK506-binding protein 5 (FKBP5) were monitored by luciferase reporter gene assay and Western blot , respectively. The bicalutamide-resistant cell line, LNCaP-DR, was constructed, and the subcellular localization of AR and AR-V7 pro-teins in LNCaP, LNCaP-siAR-V7 and LNCaP-DR cells was observed by the method of immunofluorescence .The protein in-teraction of AR-V7 and heat shock protein 90 ( HSP90) was determined by co-immunoprecipitation .RESULTS:The mR-NA level of AR-V7 in the 4 prostate cancer cell lines was significantly higher than that in normal prostate epithelial cell line RWPE-1 (P<0.05).The AR-V7 level, cell viability and cell migration rate in the cells transfected with siAR-V7 were notablely lowered compared with the NC siRNA-transfected cells (P<0.05).The cell viability was gradually decreased following with the increase in bicalutamide dose , and down-regulation of AR-V7 expression significantly enhanced the sensi-tivity to bicalutamide (P<0.05).Down-regulation of AR-V7 expression significantly inhibited AR promoter activity and reduced the protein levels of PSA and FKBP5 (P<0.05).The results of immunofluorescence observation showed that most AR and AR-V7 were mainly located in the nucleus , a few AR was located in the cytoplasm , and down-regulation of AR-V7 expression inhibited AR nuclear transport .AR was entirely located in the nucleus and the protein levels of AR-V7 was sig-nificantly increased in the bicalutamide-resistant cells .The interaction of endogenous AR-V7 with HSP90 was found in the prostate cancer cells .CONCLUSION: High AR-V7 level is found in the prostate cancer cells , and down-regulation of AR-V7 expression inhibits the cell viability and migration .High AR-V7 level is related to the bicalutamide resistance .The possible mechanism is that AR nuclear transport is mediated by the interaction of AR -V7 with HSP90 to activate AR signal pathway and regulate targeted gene transcriptional activity , thus resulting in drug resistance .

Genome-wide analysis of histone H3 lysine 4 trimethylation by ChIP-chip in rat lung fibroblast transdifferentiation / 中国药理学与毒理学杂志

OBJECTIVE To analyze trimethylation of genome-wide histone H3 lysine 4(H3K4met3) induced by silicon dioxide(SiO2)through chromatin immunoprecipitation linked to microarrays(ChIP-chip)in lung fibroblast(LF)of rats. METHODS A primary co-culture model of rat alveolar macrophages (AM)and LF in vitro. AM were exposed to 100 mg · L-1 free SiO2 for 24 h,before LF were collected and the phenotype of LF was determined after transdifferentiation by immunohistochemistry. ChIP-chip was used to profile the variations of trimethylation in H3K4 of lung fibroblasts in CpG island regions. ChIP-qPCR was used to validate the microarray results. The mRNA expression of nfib and kpna3 was analyzed by qRT-PCR. RESULTS Totally 1815 (518 increased and 1297 decreased) genes of H3K4met3 displayed significant differences in SiO2 100 mg·L-1 group compared with control group(Cy3/Cy5 value>2.0 or <0.5,NimbleScan V2.5 software). The results of ChIP-qPCR were quite consistent with those of microarray. CONCLUSION There are significant differences in methylation of genome-wide H3K4 between SiO2 100 mg·L-1 group and control group. These novel candidate genes may become potential biomarkers or new interfered targets.

Identification of the binding region for interaction of optineurin with neural retina leucine / 中华实验眼科杂志

Background Gene encoding optineurin (OPTN) is a causative gene for glaucoma and amyotrophic lateral sclerosis,with a more expression in retina.Our previous study isolated OPTN-interacting proteins and identified that the gene encode the basic leucine zipper (bZIP) transcription factor neural retina leucine (NRL) zipper,a causative gene for retinitis pigmentosa,and further study demonstrated the interaction between OPTN and NRL proteins in nuclei of cultured HeLaS3 cells.Objective This study was to determine the protein binding site of OPTN necessary for NRL binding.Methods A deletion series of OPTN-expression plasmids were constructed and co-expressed with hemagglutinin (HA)-tagged NRL in HeLaS3 cells,respectively.The cytoplasmic and nuclear fractions were used to perform co-immunoprecipitate (CoIP) and Western blot with anti-tag antibodies.Results In the nuclear fractions of cells transfected with the del1 st,del2nd or del3rd plasmid,a band of coimmunoprecipitated HA-labelled NRL (HA-NRL) was detected.However,the del4th plasmid did not produce a band.The NRL band was not found in cytoplasmic fractions from transfected cells with any of the deletion plasmids or with the whole-length OPTN plasmid.Conclusions The protein binding site of OPTN necessary for NRL binding is determined.This result demonstrates the binding of Flag-OPTN and HA-NRL in HeLaS3 cells.A series of partial-deletion OPTN plasmids demonstrated that the tail region (423-577 amino acids) of OPTN was necessary for binding with NRL.

Co-immunoprecipitation in screening proteins interacting with CXCR4 during nuclear localization in renal cell carcinoma A498 cells / 第二军医大学学报

Objective To screen for the proteins interacting with CXCR4 during nuclear localization in renal cell carcinoma (RCC) A498 cells. Methods Specific band in co-immunoprecipitation (Co-IP) experiments was sent for mass spectrometry. With the results of Co-IP experiments and mass spectrometry, the proteins interacting with CXCR4 were determined by bioinformatics analyses. Results Three specific bands were found after Co-IP with anti-CXCR4 antibody, and the results of mass spectrometry of the three specific bands showed 36 proteins possibly interacting with CXCR4. Bioinformatics analyses showed that NR1D2, c-src and HSPA8 might interact with CXCR4 and participate in CXCR4 nuclear localization. Conclusion NR1D2, c-src and HSPA8 might have participated in CXCR4 nuclear localization in RCC A498 cells.

Verification of interaction between glutamate-ammonia ligase and nuclear localization signal-retinoic acid receptor α protein inside and outside cells / 第二军医大学学报

Objective: To verify the interaction between glutamate-ammonia ligase (GLUL) and nuclear localization signal-retinoic acid receptor α (NLS-RARα) protein by yeast two-hybrid and co-immunoprecipitation method. Methods: The two plasmids expressing NLS-RARα bait-protein and GLUL protein were co-transformed into yeast AH109 to investigate the interaction in vivo. Tagged fusion protein eukaryotic expression vectors were constructed and co-transfected into HEK 293 cells. Co-immunoprecipitation was used to investigate the interaction between NLS-RARα and GLUL in vitro. Results: Positive blue clones were found in the QDO/X-α-gal plate. Eukaryotic expression vectors were co-transfected into HEK 293 cells, then HA-NLS-RARα protein was immunoprecipitated by anti-HA polyclonal antibody, and GLUL-cMyc protein expression was confirmed by Western blotting analysis using anti c-Myc monoclonal antibody. Conclusion: The interaction between NLS-RARα and GLUL has been verified by both yeast two-hybrid and co-immunoprecipitation.

Interaction between nuclear localization signal-retinoic acid receptor α and Ubiquilin 1 / 中南大学学报(医学版)

Objective To identify the interaction between nuclear localization signal-retinoic acid receptor α (NLS-RARα) and Ubiquilin 1(UBQLN1). Methods The recombination expression plasmids pGBKT7-NLS-RAR and pACT2-UBQLN1, which expressed bait-protein NLS-RARα and target protein UBQLN1 respectively, were cotransformated into yeast AH109. The interaction of the expression plasmids in the living cells was investigated by yeast two-hybrid assay. HA-tagged fusion protein (pCMV-HA-NLS-RARα) expression vector and Myc-tagged fusion protein (pCMV-Myc-JTV1) expression vector were constructed, identified, and cotransfected respectively into human embryo kidney 293 cells(HEK293). The interaction was detected by co-immunoprecipitation.Results Blue clones were found on yeast AH109 plate cotransformated with pGBKT7-NLS-RARα and pACT2-UBQLN1. Double restriction enzyme digestion showed that pCMV-HA-NLS-RARα and pCMV-Myc-JTV1 were successfully constructed. Then HA-NLS-RAR protein was immunoprecipitated by anti-HA polyclonal antibody and the expression of Myc-UBQLN1 was tested by Western blot with anti-Myc monoclonal antibody from immunoprecipitated complex. Conclusion The interaction between NLS-RARα and UBQLN1 can be verified by yeast two-hybrid assay and co-immunoprecipitation.

Interaction between fragile histamine triad and protein kinase C alpha in human non-small cell lung cancer tissues / 西安交通大学学报·英文版

Objective: To investigate the interaction between fragile histamine triad (FHIT) and protein kinase C alpha (PKCα) in human non-small cell lung cancer tissues. Methods: FHIT and PKCα double positive samples were screened by immunohistochemical staining from 13 human non-small cell lung cancer tissues. Co-immunoprecipitation was performed by using anti-FHIT and anti-PKCα. The immune precipitate was analyzed by SDS-PAGE and Western blot. Results: Immune precipitate staining detection showed that 3 samples out of the 13 cases were double positive for FHIT and PKCα. FHIT protein was present in the immune precipitate of anti-PKCα while there was PKCα in the immune precipitate of anti-FHITmAb. Conclusion: FHIT and PKCα exist as a complex in human non-small cell lung cancer tissues, which will provide a new route for studying the pathogenesis and immunotherapy of human non-small cell lung cancer.

Isolation and preliminary identification of 74×103 molecule as putative Japanese encephalitis virus receptor / 中华微生物学和免疫学杂志

Objective To isolate and identify the putative Japanese encephalitis virus (JEV) re-ceptors from C6/36 and Vero cells. Methods Molecules binding with JEV were isolated from C6/36 and Vero cells by co-immunoprecipitation (Co-IP) approach, identified by mass spectrometry, and detected by Western blot. The location of putative JEV receptor on cells membrane and the binding with JEV were ob-served by laser scanning confocal microscopy (LCM). Results Several molecules binding with JEV were isolated from C6/36 and Vero cells by Co-IP, and only one molecule was identified as heat shock cognate 70 (HSC70) by mass spectrometry. Antibody against HSC'70 was able to detect a 74 ×103 protein isolated by Co-IP from C6/36 and Vero cells membrane in Western blot assays. It was observed by LSCM that when JEV attached on the surface of C6/36 cells, JEV and HSCT0 protein were co-localization. Conclusion 74 x 103 molecular identified as HSC70 protein from C6/36 cells may be JEV receptor.

NOK Activates STAT3 Signaling by a JAK2-Dependent Mechanism / 生物化学与生物物理进展

Novel oneogene with kinase-domain (NOK) can activate multiple mitogenic signaling pathways including the janus kinases(JAK) and signal transducer and activator of transcription proteins (STAT). It was showed that NOK specifically and physicallyinteracted with STAT3 in human embryo kidney 293T (HEK293T) cells. In addition, NOK could directly interact with most of the STAT3 subdomains except coiled-coil and C-terminal domains. Removing ectodomain and transmembrane domain of NOK markedly enhanced its intermolecular interaction with STAT3. Also, NOK could co-immtmoprecipitate with JAK2. in vivo. Importantly, co-expression of NOK and JAK2 produced a synergistic effect on NOK-mediated STAT3 activation, while inactivating the kinase domain of JAK2 completely prevented this synergistic effect. Overall, the results indicated that NOK might complex with both STAT3 and JAK2 and activate STAT3 signaling by a JAK2-dependent mechanism.

Separation and identification of hsp27 interacting proteins from human hepatocellular carcinoma cells / 重庆医科大学学报

Objective:To find out heatshock protein HSP27 interactingproteins in the hepatocellularcarcinoma(HCC).Methods:HSP27 interacting proteins in the human HCC HepG2.2215 cells were seperated and identified.HSP27 binding proteins were recovered by antiHSP27 antibody immunoprecipitation with the total proteins from HCC HepG2.2215 cells.After in-solution digested and analyzed by LC-ESI-ITMS/MS,the recovered protein complexes were identified by peptide sequence tags(PST)and database searching,and were confimed by immunoprecipitation and Western blot analysis.Results:Seven HSP27 binding proteins were firstly identified by database searching,which were HSP90,GRP-78 and HSP70 of HSP70 family members,lysyl hydroxylase,Keratin 9,Keratin 10,Keratin 1.The identified seven HSP27-interacting proteins by IP coupled with MS could be classified into three functional categories based on their functions:chaperones,cytoskeleton organization,metabolic enzymes.Conclusion:HSP27 in HCC is a related proteins involved in the regulation,by interacting with some peptides,or small molecule protein to control the tumor development.