Sc 136 288

RIPA buffer (Beyotime) containing protease inhibitor was used for protein extraction from the collected tissues or cells. The protein concentration was determined by using BCA Kit (Beyotime). Then equal amounts of proteins were denatured by boiling water bath for 10 min and then separated by SDS-PAGE. The separated proteins were transferred onto 0.45 μm PVDF membranes (Millipore, Billerica, MA, USA) and then blocked with 5% non-fat milk for 1 h. Subsequently, the membranes were incubated with primary antibodies against MAPK1 (sc-136288, 1:1000 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA), c-Myc (ab39688, 1:500 dilution, Abcam, Cambridge, MA, USA), Bax (ab199677, 1:1000 dilution, Abcam) or MMP9 (ab119906, 1:1000 dilution, Abcam) at 4 °C overnight and corresponding secondary antibody at room temperature for 2 h. GAPDH (ab37168, 1:2000 dilution, Abcam) was used as an internal control. The ECL system (Beyotime) was applied to visualize the protein blots and the relative protein levels were normalized to corresponding control group.