Anti htert

As described previously [24] , more than 5×10 6 primary PTECs, hTERT-PTECs and the positive control HEp-2 cells (CLS Cat No.300397/p694_Hep-2) were harvested after twice PBS washing before the cells were extracted with total protein extraction kits (Vazyme, Cat No. E211). Cellular proteins were stored at -80°C directly after rapid liquid nitrogen frozen for 30 s. Approximately 20 µg of each total protein was resolved by 10% SDS-PAGE before being transferred to a nitrocellulose lter membrane (Thermo Fisher, Cat No.77010). The membranes were blocked with 5% nonfat milk in Tris Buffered saline (TBS), which contains 0.5% Tween 20 (TBST) and were put in a 37°C incubator with gentle shaking for complete reaction for 2 h. Then the membrane was followed by incubation with anti-hTERT (Santa Cruz Biotechnology, Cat No. sc-377511) or anti-β-actin (Bioworld, Cat No. BS6007MH) at 1:600 with 5% nonfat milk in TBST in a 37°C incubator for 1.5 h. After three times TBST washing, the membranes were incubated with secondary antibody (HRP-labeled goat anti-mouse IgG (H+L) (Boster, Cat No.BA1051) at 1:5000 with 5% nonfat milk in TBST for 1 h. Incubation with electrochemiluminescence substrate reagents (Thermo Fisher, Cat No. 32109) was conducted after three times TBST washing, before the membrane was nally observed with a ChemiDoc XRS+ system (Bio-Rad).