Rabbit anti klf4

Western blotting was carried out as described previously [26] (link). Briefly, crude proteins were extracted, resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and transferred onto a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). Membranes were blocked with 5% milk in Tris-buffered saline with Tween 20 for 2 h at 37 °C and then incubated overnight at 4 °C with the following primary antibodies: rabbit anti-KLF4 (1:400, Santa Cruz Biotechnology), rabbit anti-cleaved caspase-3 (1:500, Proteintech, Chicago, IL, USA), rabbit anti-bax (1:500, Proteintech), or mouse anti-β-actin (1:1000, Santa Cruz Biotechnology). After incubation with the appropriate secondary antibody, the immunoreactive signal of antibody–antigen pairs was visualized using the Chemiluminescence Plus Western Blot analysis kit (Santa Cruz Biotechnology). Experiments were performed at least three times.

Protein was extracted from the cells and tissues using RIPA lysis buffer [1% NP40, 0.1% sodium dodecyl sulfate (SDS), 100μg/ml phenylmethylsulfonyl fluoride, 0.5% sodium deoxycholate, in PBS] on ice. The super-natants were collected following centrifugation at 12,000 × g at 4°C for 20 min. The protein concentration was determined using a BCA protein assay kit (Bio-Rad, Shanghai, China), and whole lysates were mixed with 4X SDS loading buffer [125 mmol/l Tris-HCl, 4% SDS, 20% glycerol, 100 mmol/l dithiothreitol (DTT) and 0.2% bromophenol blue] at a ratio of 1:3. The samples were heated at 100°C for 5 min and were separated on SDS-polyacrylamide gels. The separated proteins were then transferred onto PVDF membranes (Millipore, Bedford, MA, USA). The membranes were first probed with a primary antibody. After blocking with 5% skim milk for 2 h, the membranes incubated with primary antibodies [rabbit anti-KLF4 (1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA). The secondary antibody was mouse anti-GAPDH (1:3000, Santa Cruz Biotechnology).