Standard bradford assay

Upon harvesting in ice-cold RIPA Lysis and Extraction buffer (ThermoFisher #89900) fortified with Halt Protease and Inhibitor Cocktail (ThermoFisher #78440), samples were finely minced, homogenized, and stored at −80° C. Prior to electrophoresis, tissues were thawed at 0° C and homogenized with a motorized Teflon pestle, followed by determination of total protein concentration using a Standard Bradford Assay (Bio-Rad #500–0006) and dilution in Laemmli Sample Buffer (Bio-Rad #161–0747) to a concentration of 2.5 mg/mL. For electrophoresis, 20 μg of each sample were loaded into each lane of a precast Bio-Rad 4–20% acrylamide gel, and separated proteins were transferred (60 min at 100 V) onto 0.45μm nitrocellulose membrane (Bio-Rad #162–0145). The blots were blocked with 5% non-fat dry milk/10 mM Tris-HCl (pH 7.6)/150 mM NaCl/Tween-20 (5% NFDM/TBST) or 5% BSA in TBST. Primary and secondary antibodies and dilutions are listed in Supplemental Table 2. Blots were reacted with primary antibody in 5% NFDM/TBST or 5% BSA blocking buffer overnight at 4°C. Secondary antibodies were diluted in 5% NFDM/TBST and applied for 60 minutes at RT. Reacted blots were covered with horseradish peroxidase substrate (ThermoFisher #34580) for 5 min at room temperature, followed by chemiluminescence imaging and densitometry using Bio-Rad ChemiDoc and ImageJ software, respectively.