Quantification of MAPK Signaling Proteins

Total cellular proteins were extracted from HCEC-B4G12 cells treated with Na-Mt, and protein concentrations determined by using the BCA Protein Assay Kit (Beyotime). The primary antibodies used for this were p38, phospho-p38 MAPK (Thr180/Tyr182), ERK1/2, phospho-ERK1/2 (Thr202/Tyr204), JNK, and p-JNK (Thr183/Tyr185) (Cell Signaling Technology; Danvers, MA, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Proteintech, 60,004-1-lg) served as the loading control for total cellular proteins. Western blot analysis was performed from three independent experiments for each treatment. To quantify the intensity of the resulting bands, Image J software was used (v1.53c).