Rabbit monoclonal anti gapdh

Cells were lysed with RIPA buffer (Pierce, Rockford, IL, USA) The BCA Protein Assay Reagent (Pierce) was used to quantify the protein concentration of lysates. Equal amounts of protein were resolved by 8–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Then the proteins were transferred to the polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). After blocking for 4 h at room temperature in 10% nonfat dry milk in TBST containing 0.05% Tween-20, the membranes were incubated with primary antibodies overnight at 4 °C and then after washes using TBST, it was incubated with HRP-conjugated secondary antibodies for 2 h at room temperature.
Primary antibodies included rabbit anti-Klf4 (Abcam, Cambridge, UK, 1:1000) Rabbit monoclonal anti-GAPDH (Sigma, 1:5000) rabbit anti-Nanog (CST, Danvers, MA, USA), Anti-ERK1/2 rabbit polyclonal antibody (sangon biotech, Shanghai, China, 1:1000), Anti-ERK1/2(Thr202/Tyr204)) rabbit polyclonal antibody (sangon biotech, 1:1000), Rabbit Polyclonal anti-ABHD2 (Proteintech, Rosemont, IL, USA, 1:1000) and anti-rabbit/mouse horseradish peroxidase-conjugated secondary antibody were obtained from the Beyotime Institute of Biotechnology (Jiangsu, China ).

Total protein of J1 mESCs or F9 EC cells was extracted, and its concentration was determined using the bicinchoninic acid protein assay (Beyotime). Equal amounts of protein were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Then the proteins were transferred to the polyvinylidene fluoride membranes (Millipore). After blocking for 3 h at room temperature in 10% nonfat dry milk in TBST containing 0.05% Tween-20, the membranes were incubated with primary antibodies overnight at 4°C and then after washes were incubated with HRP-conjugated secondary antibodies for 2 h at room temperature. Peroxidase activity was detected through autography using SuperSignal west pico substrate (Pierce/Thermo Scientific, Rockford, IL, USA). Primary antibodies included rabbit anti-Klf4 (Abcam, Cambridge, UK, 1:1000) and anti-β-catenin (Santa Cruz, CA, USA, 1:1000). Rabbit monoclonal anti-PCNA (Abcam. 1:1000). Rabbit monoclonal anti-GAPDH (Sigma. 1:5000).