Precision plus protein prestained standard in all blue

Protein was extracted by homogenizing tissues in radioimmunoprecipitation assay (RIPA) buffer containing a protease inhibitor to preserve protein integrity (cOmplete^TM, Mini, EDTA-free Protease Inhibitor Cocktail, Sigma-Aldrich, Castle Hill, NSW, Australia) [29 (link)]. Tissues were sonicated and then cleared by centrifugation at 12,000× g for 10 min. Protein quantification was determined with bicinchoninic acid assay (Pierce BCA Protein Assay Kit, ThermoFisher Scientific, North Ryde, NSW, Australia). Equal amounts of protein (30 μg) was separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using 4–20% Mini-PROTEAN TGX Stain-Free Gels (15 well, BioRad, South Granville, NSW, Australia). The Precision Plus Protein Prestained Standard in All Blue (BioRad, South Granville, NSW, Australia) was included for comparison. Transfer to a PVDF membrane was performed using the semi-dry method (BioRad Trans-Blot Turbo Transfer System). Incubation with primary antibodies was performed overnight in 5% skim milk in TBST blocking solution at 4 °C. Antibodies and dilutions are summarized in Table 2. Western blots were visualized using chemiluminescence BioRad Clarity Western ECL Blotting Substrate Solution. Images were acquired using the BioRad ChemiDoc MP System. Images were analysed using Fiji ImageJ and ratios normalized to GAPDH which was used as a loading control.

Protein was extracted by homogenizing tissues in radioimmunoprecipitation assay (RIPA) buffer as previously described, and quantified using the bicinchoninic acid assay (Pierce BCA Protein Assay Kit, ThermoFisher Scientific, VIC, Australia). Next, 30 μg of protein was separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using 4–20% Mini-PROTEAN TGX Stain-Free Gels (15 well, BioRad, VIC, Australia). The Precision Plus Protein Prestained Standard in All Blue (BioRad, VIC, Australia) was included for comparison. Transfer to a PVDF membrane was performed using the semi-dry method (BioRad Trans-Blot Turbo Transfer System). Primary antibodies were incubated overnight in 5% skim milk in TBST blocking solution at 4 °C. Antibodies and dilutions are summarized in Table 3. Membranes were incubated in secondary antibody 1 hr at RT. Western blots were visualized using chemiluminescence BioRad Clarity Western ECL Blotting Substrate Solution. Images were acquired using the BioRad ChemiDoc MP System. Images were analysed using Fiji ImageJ and ratios normalized to GAPDH which was used as a loading control.