Quantitative Protein Expression Analysis

Western blot analyses of whole-cell protein lysates (RIPA lysis buffer supplemented with protease and phosphatase inhibitors, Thermo Fisher Scientific) were performed as described^{12 (link)}. Protein concentrations were assayed using a BCA protein assay Kit (Pierce). Equal amounts of protein were separated by SDS-PAGE using 4–20% gradient Tris-glycine gels (Bio-Rad) and transferred to nitrocellulose membranes (Bio-Rad). After incubation with primary and secondary antibodies, the proteins were detected using Western Bright Quantum chemiluminescence detection system (Advansta). Scanning of gels was performed using Epson Perfection 4490 Photo Scanner, and quantification using Fiji ImageJ. In some cases, Odyssey imaging (Li-Cor Biosciences) was used and the relative band densities quantified with Image Studio Lite Ver 5.2 software (Li-Cor Biosciences).