Ab111135

Cells were homogenized in RIPA lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) containing with freshly added protease inhibitors and phosphatase inhibitor. Protein was analyzed with a bicinchoninic acid assay (Beyotime Biotechnology, Shanghai, China) to quantify protein concentration. A total of 30 µg of total protein was loaded per lane. Proteins were resolved by 10% SDS-PAGE and transferred to PVDF membranes. 3% BSA (Solarbio, Beijing, China) was administrated at room temperature for 90 min to block nonspecific binding sites. The anti-AP1M1 rabbit polyclonal antibodies (ab111135) was purchased from Abcam (Cambridge, UK), the β-actin antibody (AC026) was purchased from Abclonal (Wuhan China), the antibodies for total JNK (#9252), AKT (#4691) and phosphorylated JNK (#4668) and AKT (#4060) were purchased from CST (Boston, MA, USA). The final dilution of AP1M1 antibodies was 1:500, β-actin antibody was 1:5,000, the others were 1:1,000. After incubation with the primary antibodies overnight at 4°C, the PVDF membranes were washed with TBST buffer thrice and incubated with 1:1,000 diluted HRP-linked secondary antibody (#7074) at room temperature for 60 min. The labeled bands were detected with ECL Plus Western blotting detection kit (Beyotime Institute of Biotechnology).