Anti-radiation damage effect of polyethylenimine as a toll-like receptor 5 targeted agonist

Hu, Zhiqiang; Xing, Yaling; Qian, Yuanyu; Chen, Xiaojuan; Tu, Jian; Ren, Lening; Wang, Kai; Chen, Zhongbin

HEK 293T cells were seeded onto 24-well plates and incubated at 37°C for 18 h before transfection. The cells were subsequently transfected with pcDNA3.1-V5/HisB-hTLR5 or empty vector using Lipofectamine TM 2000 reagent according to manufacturer's instructions. Forty-eight hours after transfection, cells were lysed in buffer containing 0.5% TritonX-100, 150 mM NaCl, 12.5 mM β-glycerolphosphate, 1.5 mM MgCl 2 , 2 mM EGTA, 10 mM NaF, 1 mM Na 3 VO 4 and 2 mM DTT plus protease inhibitor cocktail (Sigma). The cell extracts were spun down at 5000 × g at 4°C for 10 min. Protein concentration of lysate was determined using BCA Protein Assay kit (Bio-Rad). Protein extracts in 30 µl of 2 × sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer were boiled for 10 min. Samples were separated on SDS-PAGE gel and transferred to a polyvinylidene difluoride (PVDF) membrane. Blots were incubated with V5 antibody (MBL, Japan). After washing three times in 1 × TBS-T buffer, blots were subsequently incubated with goat-anti-rabbit-HRP (Beyotime, China). Antibody-antigen reactions were detected using Western Lighting Plus-ECL chemiluminescence reagents (Biomed, China).