Western Blot Analysis of Protein Targets

Protein extracts from cells were obtained as previously reported [17 (link)]. The protein concentrations were determined using the Bradford assay [52 (link)]. Protein extracts (25 μg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA), incubated with anti-Twist1 (SC-81417, Santa Cruz Biotechnology), anti-IL-17RA (FAB177P, R&D Systems, Minneapolis, MI, USA), and anti-Act1 (SC-100647, Santa Cruz Biotechnology) antibodies at a 1:500 dilution at 4 °C overnight, followed by incubation with the appropriate secondary antibody at a 1:2000 dilution at room temperature for 2 h. Anti-GAPDH (SC-47724, Santa Cruz Biotechnology) at a 1:2000 dilution was used as loading control. Antibody binding was detected using enhanced chemiluminescence with Pierce Plus Western Blotting Substrate (Thermo Scientific). Images were obtained using iBright FL1500 Imaging System instrument (Thermo Scientific). Densitometric analysis was conducted using ImageJ 1.53k (NIH). Values obtained for GAPDH staining was used to normalize the densitometry values to the probed targets.