Trans blot semi dry electrophoretic transfer cell system

Soluble protein extraction from HEK293T cells was performed using Cytobuster Protein Extraction Reagent (Fisher Scientific) according to the manufacturer’s instructions. Protein extraction solutions were supplemented with 1X Halt Protease Inhibitor (Fisher Scientific) and 1X Halt Phosphatase Inhibitor (Fisher Scientific) Cocktails. Protein concentrations were determined using A280 on a NanoDrop 1,000 Spectrophotometer and subsequently analyzed by Western immunoblotting. Equivalent amounts of protein (40 µg) were separated by 10% SDS-PAGE and transferred to PVDF membranes (Millipore Sigma) using the Trans-Blot Semi-Dry Electrophoretic Transfer Cell System (Bio-Rad); Ponceau S staining was used to assess the equivalency of protein loading after SDS-PAGE transfer. Membranes were washed with 1.0M Tris-buffered saline (TBS), pH 7.4, blocked with 5% Bovine Serum Albumin (BSA) in TBS for 1 hour at room temperature, and then incubated with primary antibodies (Key Resources Table) in 5% BSA in TBS-T (0.1% Tween 20) overnight at 4°C. Following TBS-T washes, membranes were incubated with peroxidase-conjugated secondary antibodies in TBS-T (Key Resources Table) and protein bands were detected using Clarity Western ECL Substrate (Bio-Rad) and visualized using the ChemiDoc Imaging System (Bio-Rad).

Nuclear/cytoplasmic subcellular fractionation was performed using NE-PER Nuclear and Cytoplasmic Extraction Reagents Kit (Thermo Fisher Scientific, Burlington, ON, Canada) according to manufacturer′s instructions. Cytoplasmic protein concentrations for individual fractions were determined using A280 on a NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific, Burlington, ON, Canada) and subsequently analyzed by Western immunoblotting. Equivalent amounts of protein (40 µg) were separated by 10% SDS-PAGE and transferred to PVDF membranes (Millipore Sigma, Oakville, ON, Canada) using the Trans-Blot Semi-Dry Electrophoretic Transfer Cell System (Bio-Rad, Mississauga, ON, Canada); Ponceau S staining was used to assess the equivalency of protein loading after SDS-PAGE transfer. Membranes were washed with 1.0 M Tris-buffered saline (TBS), pH 7.4, blocked with 5% Bovine Serum Albumin (BSA) in TBS for 60 min at room temperature, and then incubated with primary antibodies (Table 1) in 5% BSA in TBS-T (0.1% Tween 20) overnight at 4 °C. Following TBS-T washes, membranes were incubated with peroxidase-conjugated secondary antibodies (Table 1) and protein bands were detected using Clarity Western ECL Substrate (Bio-Rad, Mississauga, ON, Canada) and visualized using the ChemiDoc Imaging System (Bio-Rad, Mississauga, ON, Canada).