Quantitative Western Blot Analysis

Western blotting experiments were carried out according to our previous study [26 (link), 27 (link)]. Briefly, cells were lysed in RIPA buffer (1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 10 ng/mL PMSF, 0.03% aprotinin, and 1 μM sodium orthovanadate) on ice for 30 min. Protein concentration of samples was measured with the bicinchoninic acid (BCA) assay method. Proteins were separated on 8–12% SDS-PAGE gels and transferred to nitrocellulose membrane (Millipore, USA). Membranes were blocked with 5% BSA in TBS with 0.1% tween-20 and incubated with primary antibodies as follows: anti-TRPM7 (1 : 1000, Abcam, #ab85016, USA), anti-p-Akt-Ser473 (1 : 1000, Cell Signaling Technology, #4060, Inc., USA), anti-Akt (1 : 1000, Cell Signaling Technology, #2920 Inc., USA), phospho-p44/42 MAPK (p-ERK1/2, 1 : 1000, Cell Signaling Technology, #8544, Inc., USA), anti-ERK1/2 (1 : 1000, Cell Signaling Technology, #4696, Inc., USA), and anti-β-actin (1 : 1000, Cell Signaling Technology, #3700, Inc., USA) antibodies followed by incubation with corresponding horseradish peroxidase-conjugated secondary antibodies were used against each primary antibody. Bands were developed with a chemiluminescence reagent system (Beyotime, China).