Mouse anti gapdh monoclonal antibody

Cells were lysed with radio immuno-precipitation assay buffer (Beyotime) containing a protease inhibitor cocktail (cOmplete™, Mini, EDTA-free, EDTA-free Protease Inhibitor Cocktail; Roche). The cell lysate was incubated on ice for 15min and centrifuged at 4 ºC for 20 min at 14000g. The protein concentration of the supernatant was measured using an enhanced BCA protein assay kit Enhanced BCA Protein Assay Kit Enhanced BCA Protein Assay Kit Enhanced BCA Protein Assay Kit (Beyotime). The denatured proteins were resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions and transferred to polyvinylidenefluoride membranes (GE Healthcare). The membranes were incubated with primary antibodies at 4 °C overnight. After washing in TBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies and the immunosignals were visualized using the Super ECL Detection Reagent (yeasen, Shanghai). Primary antibodies were: rabbit anti-METTL3 (1:2000, Abcam), rabbit anti-METTL14 (1:2000, Sigma), mouse anti-GAPDH monoclonal antibody (1:5000, ZSGB-BIO).

Recombinant IL-8 was purchased from PeproTech, INC. Polyclonal antibodies against TMEM98 were purchased from Abcam. Alpha tubulin was purchased from Proteintech. Mouse anti-GAPDH monoclonal antibody was purchased from ZSGB-BIO. Monopoly antibodies against p-AKT (S473), p-AKT (T308), t-AKT, p-PI3K p85 (Y607), p-GSK3β (S9) and p-c-Raf (S259) were purchased from Cell Signaling Technology. The antibodies against p-ERK1/2, ERK and Cyclin D1 were purchased from Bioworld. The antibodies against of PI3K and p-PI3K were purchased from abcam. SC79 and BEZ235 were purchased from Selleck.cn. Fluorescein isothiocyanate (FITC)-conjugated anti-α-smooth muscle actin antibody (F3777) and phalloidin-tetramethyl rhodamine B isothiocyanate (P1951) were obtained from Sigma-Aldrich (St Louis, MO, USA).