Stain free tgx gels

Frozen colon tissue samples were lysed in RIPA Buffer and the total protein concentration was quantified using a BCA assay kit as per manufacturer’s protocol (Thermo Scientific). Lysates were obtained from three independent biological samples per group and pooled into a single sample for analysis. Polyacrylamide gel electrophoresis was performed to separate proteins using 4–20% stain-free TGX gels (Bio-Rad). Proteins were visualised by UV-induced fluorescence using a Chemidoc imaging system (Bio-Rad) to verify equal loading of samples. The most abundant protein band in each loading control sample was used for quantification. Proteins were transferred to nitrocellulose membranes in a trans-blot turbo transfer system (Bio-Rad). Membranes were blocked for 30 min using 5% skimmed milk in PBST and then probed with rat anti-GFAP primary antibody (1:2000, cat # 13-0300, ThermoFisher) overnight at 4 °C, followed by incubation with HRP-conjugated goat anti-rat secondary antibody (1:5000, cat # 31460, ThermoFisher) for 2 h at room temperature and visualisation using enhanced chemiluminescence (ECL kit, GE Healthcare Life Sciences). Data were analysed using the gel analysis package in FIJI^{66 (link)}.

Protein samples from skeletal muscle were prepared by first snap freezing dissected tissues in liquid nitrogen, then homogenizing tissues in RIPA buffer supplemented with protease and phosphatase inhibitors (Thermo Fisher Scientific) using a Qiagen Tissuelyser LT and Qiashredder column. Soluble protein was quantified by Qubit Protein Assay and prepared according to manufacturer instructions (Thermo Fisher Scientific). Proteins were separated on 4–20% Stain-Free TGX gels (Bio-Rad), transferred to PVDF, and blocked in 5% non-fat dry milk in TBST (1X TBS with 0.05% Tween-20) for 1 h at RT. Membranes were incubated with primary antibody recognizing human and mouse AR (Abcam #ab108341) for 48 h, washed, then incubated with anti-rabbit secondary antibody conjugated to horseradish peroxidase for 1 h. Signal was detected with SuperSignal West Dura (Thermo Fisher Scientific) and imaged on a Bio-Rad ChemiDoc system. Densitometry analysis was performed using Image Lab software and normalized to total protein as detected by stain-free technology (Bio-Rad).