Anti p erk1 2

Western blotting analyses were performed as previously described using 30μg lysates from fresh tissues and cells [6 (link)]. Proteins were visualized by exposing Chemiluminescent HRP Substrate (Millipore, Billerica, MA, USA) by protein imprinting imaging system (Tanon5200, China). Antibodies against human GDF15, AKT1, Erk1/2, GAPDH were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-PI3K (p-p110a) was from Abcam Technology (Cambridge, MA, USA). Anti-p-AKT was purchased from CST (Littleton, CO, USA). Anti-Ras and Anti-Ras-GTP was from NewEast Biosciences (WuHan, CN). Anti-p-Erk1/2, anti-ErbB2, anti-p-ErbB2, anti-FOXO1 were from Abclonal Technology (Boston, MA, USA).

Cells were lysed with RIPA Lysis Buffer with PMSF (ST506, Beyotime Institute of Biotechnology). Proteins were quantified by BCA Protein Assay Kit (P0010, Beyotime Institute of Biotechnology). 20–80 μg of total protein was loaded onto SDS-PAGE and subsequently transferred to PVDF membranes (162-0177, Bio-Rad, Richmond, CA). After blocking with 5% nonfat milk or bovine serum albumin (BSA) in PBST, membranes were incubated with the following primary antibodies at the suggested dilutions: anti-CDKN1A/p21 (A1483, ABclonal, Cambridge, MA), anti-TP53/p53 (sc-6243, Santa Cruz Biotechnology, Santa Cruz, CA), anti-ERK1/2 (A0229, ABclonal), anti-p-ERK1/2 (AP0472, ABclonal), anti-p-Histone H2A.X (S139) (AP0099, ABclonal), anti-p-ATM (10H11.E12) (sc-47739, Santa Cruz Biotechnology), anti-p-CHK1 (Ser345) (sc-17922, Santa Cruz Biotechnology), anti-p-CHK2 (Thr68) (sc-16297-R, Santa Cruz Biotechnology), anti-PUMA (sc-374223, Santa Cruz Biotechnology), anti-TICRR (NBP2-41283, Novus Biologicals, Littleton, CO), anti-cyclin D1 (2922S, Cell Signaling Technology, Danver, MA) and anti-ACTB (AA128, Beyotime Institute of Biotechnology). Primary antibodies were detected with HRP-labeled goat anti-rabbit (A0208, Beyotime Institute of Biotechnology) or anti-mouse IgG (H+L) (A0216, Beyotime Institute of Biotechnology).