ABSTRACT
Zwitterionic sulfobetaine-based monolithic stationary phases have attracted increasing attention for their use in hydrophilic interaction chromatography.In this study,a novel hydrophilic polymeric monolith was fabricated through photo-initiated copolymerization of 3-(3-vinyl-1-imidazolio)-l-propanesulfonate(SBVI)with pentaerythritol triacrylate using methanol and tetrahydrofuran as the porogenic system.Notably,the duration for the preparation of this novel monolith was as little as 5 min,which was significantly shorter than that required for previously reported sulfobetaine-based monoliths prepared via conventional thermally initiated copolymerization.Moreover,these monoliths showed good morphology,permeability,porosity(62.4％),mechanical strength(over 15 MPa),column efficiency(51,230 plates/m),and reproducibility(relative standard deviations for all analytes were lower than 4.6％).Mechanistic studies indicated that strong hydrophilic and negative electrostatic interactions might be responsible for the retention of polar analytes on the zwitterionic SBVI-based monolith.In particular,the resulting monolith exhibited good anti-protein adhesion ability and low nonspecific protein adsorption.These excellent features seem to favor its application in bioanalysis.Therefore,the novel zwitterionic sulfobetaine-based monolith was successfully employed for the highly selective separation of small bioactive compounds and the efficient enrichment of N-glycopeptides from complex samples.In this study,we prepared a novel zwitterionic sulfobetaine-based monolith with good performance and developed a simpler and faster method for preparation of zwitterionic monoliths.
ABSTRACT
Aim Toconstructthreeplasmidsincluding Smad3 WT,Smad3 EPSM and Smad3 3S-A stable transfection in HepG2 cell lines to investigate phospho-domains of Snad3(pSmad3C or pSmad3L),their pro-tein expression and roles in HepG2 cell proliferation, apoptosisandcellcycle.Methods Threeplasmidsin-cluding Smad3 WT (Carry the wild Smad3 gene ), Smad3 EPSM(Carry the mutated phosphorylation site in linker region of Smad3 gene)and Smad3 3S-A(Car-ry the mutated phosphorylation site in C-terminal of Smad3 gene)were respectively transfected into HepG2 cells by using a liposome transfection reagent.Verifi-cation of positive cells was done by screening with G418 via co-culture.Transfection efficiency was deter-mined by Western blot.Cell proliferation was induced by exogenous TGF-β1 in the respective stably transfect-ed HepG2 cell lines.Cell proliferation was monitored by MTT.Cell cycle and apoptosis were determined by flowcytometry(FCM).Results Therewaselevated protein expression of the respective phospho-domain sites in the stably transfected HepG2 cells for Smad3 WT(C-terminus and Linker),Smad3 EPSM(C-termi-nus)and Smad3 3S-A(Linker),which indicated suc-cessful stable transfection of HepG2 cell lines.The re-sults from MTT experiment showed that TGF-β1 could induce proliferation of HepG2 cells with or without the transfection of Smad3 WT,Smad3 EPSM and Smad3 3S-A plasmids,meanwhile transfected Smad3 EPSM plasmids could significantly inhibit proliferation of HepG2 cells induced by TGF-β1 , and transfected Smad3 3S-A plasmids accelerate proliferation of HepG2 cells induced by TGF-β1 .Cell cycle analysis showed that the G0/G1 phase of HepG2 cells with stable trans-fection of Smad3 EPSM plasmid increased compared with HepG2 cells with or without stable transfection of Smad3 WT plasmid,meanwhile the G2/M phase of HepG2 cells with stable transfection of Smad3 3 S-A plasmid increased.Compared with Smad3 WT trans-fected cells, apoptosis in Smad3 EPSM transfected cells was markedly increased,while that of Smad3 3S-Atransfectedcellsdecreased.Conclusions Thethree plasmids of Smad3 WT,Smad3 EPSM and Smad3 3S-A stably transfected HepG2 cell lines have been suc-cessively constructed.The construction of three plas-mids transfected HepG2 cell lines provides the research foundation for studying medical as well as possible reg-ulatory mechanism of pSmad3 C/pSmad3 L.