Quantitative Western Blot Analysis

Cells or tissue samples were lysed in RIPA lysis buffer (Tian P. et al., 2017 (link)) added protease inhibitor (P8340, Sigma, St. Louis, MO, United States) for 30 min on ice, and then centrifuged at 12, 000 rpm for 15 min at 4°C. The supernatant was collected and measured to calculate protein concentration using a BCA protein assay kit (23225, Thermo Fisher Scientific, Waltham, PA, United States). After denaturation, 80 μg protein was electrophoresed in a 10% SDS-PAGE, and then transferred onto a nitrocellulose membrane. The membrane was blocked with 5% skimmed milk powder in TBST (Tris buffer with 0.1% Tween, pH 7.6) for 2 h and then incubated at 4°C overnight with primary antibodies: DMT1 (ab55735, Abcam, 1:500) or β-actin (BS6007M, Bioworld, 1:10000). A secondary HRP-conjugated antibody (BS12478, Bioworld, 1:10000) was used to incubate the membrane for 2 h prior to chemiluminescent detection. The signals were determined using a chemiluminescent substrate (ECL) kit (NCI4106, Thermo Fisher Scientific, Waltham, PA, United States). Then, protein intensities were quantified by the VersaDoc MP 4000 system (Bio-Rad, California, CA, United States).