Mini protean gel casting system

Tissue samples were homogenized on dry ice using a lysis buffer made of 50 mmol/L Tris-HCl, 150 mmol/L NaCl, 10% glycerol, and 0.5% Triton X-100 containing a cocktail of protease inhibitors (Roche, catalog no. 04693132001) and phosphatase inhibitor cocktail (bimake.com, catalog no. B15001). Protein concentration was quantified by Bradford assay using the Coomassie Plus Protein Assay kit (Thermo Fisher Scientific, catalog no. 23236). Samples were electrophoresed on 7.5%–15% SDS-PAGE gels using the Bio-Rad Mini-PROTEAN gel casting system. Samples were transferred to nitrocellulose membranes, blocked with 5% w/v BSA in Tris-buffered saline with Tween 20 (TBST) and then incubated with primary antibodies at a 1:1,000–1:2,000 dilution in TBST overnight at 4°C. Membranes were washed and then incubated with HRP-conjugated secondary antibodies at a 1:3,000–1:5,000 dilution in TBST overnight at 4°C. Membranes were washed and then visualized using SuperSignal West Pico (Thermo Fisher Scientific, catalog no. 34580), or SuperSignal West Femto (Thermo Fisher Scientific, catalog no. 34095) Chemiluminescent Substrate as necessary.

Tissue samples were homogenized on dry ice using a lysis buffer made of 50 mM Tris-HCl, 150 mM NaCl, 10% glycerol, and 0.5% Triton X-100 containing a cocktail of protease inhibitors (Roche, catalog 04693132001). Protein concentration was quantified by Bradford assay using the Coomassie Plus Protein Assay kit (Thermo Fisher Scientific, catalog 23236). Samples were electrophoresed on 7.5%–15% SDS-PAGE gels using the Bio-Rad Mini-PROTEAN gel casting system. Samples were transferred to nitrocellulose membranes, blocked with 5% w/v BSA in TBST, and then incubated with primary antibodies at a 1:1,000–1:2,000 dilution in TBST overnight at 4°C. Membranes were washed and then incubated with HRP-conjugated secondary antibodies at a 1:3,000–1:5,000 dilution in TBST overnight at 4°C. Membranes were washed and then visualized using SuperSignal West Pico (Thermo Fisher Scientific, catalog 34580), and SuperSignal West Femto (Thermo Fisher Scientific, catalog 34095) Chemiluminescent Substrate as necessary.

Tissue samples were homogenized on dry ice using a lysis buffer made of 50 mM Tris-HCl, 150 mM NaCl, 10% glycerol, and 0.5% Triton X-100 containing a cocktail of protease inhibitors (Roche, catalog 04693132001). Protein concentration was quantified by Bradford assay using the Coomassie Plus Protein Assay kit (Thermo Fisher Scientific, catalog 23236). Samples were electrophoresed on 7.5%–15% SDS-PAGE gels using the Bio-Rad Mini-PROTEAN gel casting system. Samples were transferred to nitrocellulose membranes, blocked with 5% w/v BSA in TBST, and then incubated with primary antibodies at a 1:1,000–1:2,000 dilution in TBST overnight at 4°C. Membranes were washed and then incubated with HRP-conjugated secondary antibodies at a 1:3,000–1:5,000 dilution in TBST overnight at 4°C. Membranes were washed and then visualized using SuperSignal West Pico (Thermo Fisher Scientific, catalog 34580), and SuperSignal West Femto (Thermo Fisher Scientific, catalog 34095) Chemiluminescent Substrate as necessary.