Western Blot Analysis of Protein Expression

Details of Western blotting were previously described^{18 (link)}. Cells at 80–90% confluence were lysed on ice in radioimmunoprecipitation assay buffer (RIPA; keygen biotech, China) containing PMFS complete protease inhibitor cocktail (keygen biotech, China). Protein concentration was determined by the BCA assay (keygen biotech, China). Equal protein samples (10 μg) were separated on 12% sodium dodecyl sulfate (SDS)/polyacrylamide gels, and transferred onto 0.45 µm polyvinylidene difluoride (PVDF) membranes (Immobilon-P; Millipore, Bedford, MA, USA). The antibodies used were as follow: anti-IMPA2 rabbit monoclonal antibody (1:1000; GeneCopoeis, USA); anti-ERK (1:10,000, Abcam, UK), anti-p38α (1:500, BBI China), anti-JNK1/2/3 (1:500, BBI China), p-ERK (1:500, Cell Signaling, USA), p-p38α (p-Thr180/Tyr182, 1:500, BBI China), and p-JNK1/2/3 (p-Th183/Ty185, 1:500, BBI China). Horseradish peroxidase (HRP)-conjugated goat anti‑rabbit immunoglobulin G (1:1000; BBI China) was used as second antibody and anti-GAPDH mouse monoclonal antibody (1:5000; BBI China) as a loading control. The final protein expression was detected by enhanced chemiluminescence (Bio-rad, Berkeley, CA, USA) according to the manufacturer’s suggested protocols. The band quantification was conducted using ImageJ (National Institutes of Health, Bethesda, MA, USA).