Optimized Western Blotting Protocol for Quantitative Protein Analysis

Western blot analysis was performed following our published protocol (11 (link)) with minor modification. Briefly, cells were lysed with the Pierce RIPA lysis buffer (89901; Thermo Fisher Scientific) containing Halt Protease Inhibitor Cocktail (87785; Thermo Fisher Scientific). Total protein concentration was determined using the DC Protein Assay Kit (5000111; Bio-Rad). The cell lysates were solubilized in Pierce Lane Marker Non-Reducing Sample Buffer (39001; Thermo Fisher Scientific) and heated at 95°C for 10 min after SDS–PAGE and Western blotting. The information for the antibodies used are available in Table S3. Specific protein bands on Western blots were quantified by the ImageJ 64 software (https://imagej.nih.gov/ij/) using Gel analyzer script. Densitometry data were normalized to loading control (GAPDH or β-actin) and then to a basal condition (e.g., vehicle and/or scrambled sequence siRNA).

Table S3 Antibodies for Western blotting.