ABSTRACT
One pair of primers were designed and synthesized based on the cDNA sequence encoding Homo sapiens poly (ADP-ribose) polymerase family, member 10 (PARP10) reported on the GenBank. The cDNA sequence encoding PARP10 was cloned from 293FT cell by RT-PCR. Then the RT-PCR product was cloned into pCMV-Myc and pEGFP-C1 plasmids. The interaction between PARP10 and beta-actin was identified through immuno-precipitation and laser confocal microscopy. Extensive expression of PARP10 in mouse tissues was confirmed by RT-PCR. Besides, Western blotting analysis indicated that cell injury caused by UV treatment could promote the expression of PARP10. The results in this paper would benefit further study of PARP10.
Subject(s)
Animals , Humans , Mice , Actins , Metabolism , Poly(ADP-ribose) Polymerases , Metabolism , Radiation Effects , Protein Interaction Domains and Motifs , Proto-Oncogene Proteins , Metabolism , Radiation Effects , Stress, Physiological , Radiation Effects , Tissue Distribution , Ultraviolet RaysABSTRACT
hUBEW, a newly identified class I ubiquitin conjugating enzyme, probably plays an important role in tumorigenesis and DNA repair processes. RNA interference (RNAi) is a process in cells to degrade specific homologous mRNA by forming duplex RNA and has been developed into a powerful tool to study gene functions. In this study, the H1-U6 dual promoter RNAi plasmid was constructed and the target sequence for hUbe2w could be transcribed from both strands and form a double stranded RNA with two 5'Uridine overhangs, which closely resembles endogenous functional siRNA. The hUbe2w cDNA was amplified from reverse transcription of the 293FT total RNA by RT-PCR, and then cloned into the pGL3-Control, pCMV-myc and pDsRed-express-C1 plasmids respectively, which were selected as report vectors to detect the RNAi effects. The plasmids were co-transfected into HEK293FT cells, and then the luciferase activity and hUBE2W protein expression were measured respectively. The Resulted reduction of mRNA and protein level demonstrate that the targets of 125 and 259 could significantly inhibit the hUbe2w expression.
Subject(s)
Humans , Base Sequence , Molecular Sequence Data , Plasmids , Genetics , Promoter Regions, Genetic , Genetics , RNA Interference , RNA, Messenger , Genetics , RNA, Small Interfering , Genetics , Physiology , RNA, Small Nuclear , Genetics , Ubiquitin-Conjugating Enzymes , Genetics , MetabolismABSTRACT
Ubiquitin conjugating enzyme functions as the second enzyme required for protein ubiquitination and plays an important role in ubiquitin transferring and substrate specific recognition. UBE2W, a newly described member of E2 family, was formerly reported probably involving in phototransduction or retinal degeneration in Drosophila. In this study, we report that murine UBE2W harbors a typical UBC domain and is highly conserved in different vertebrate homologues. GST-tagged UBE2W was expressed in E. coli BL21 (DE3) and purified with GST affinity chromatography. Using this antigen, we generated and further separated rabbit polyclonal antibody of UBE2W, of which the activity and specificity were confirmed by immunoblotting of transiently expressed myc-UBE2W fusion protein. Wide expression of UBE2W was found in brain, muscle, heart, lung, liver, spleen, kidney and testis of mouse with the generated antibody, indicating the functional importance of this novel protein. Furthermore, the UBE2W highly expression was confined to the adult testis and was developmental stage-specific.