Goat anti rabbit or anti mouse igg hrp secondary antibody

Cells were lysed with 1X cell lysis buffer (Cell Signaling Technology, 9803) containing protease inhibitor (Sigma-Aldrich, P8340) on ice for 10 min, homogenized by passing through a 21-gauge needle, and centrifuged at 14,000 x g for 15 min at 4°C to pellet the cell debris. Protein was quantified using the BCA assay (Thermo Fisher Scientific, 23225) and 20 μg of each sample was resolved on 4%–12% Criterion XT Bis-Tris gels (Bio-Rad, 3450124) in XT MES running buffer (Bio-Rad, 1610789) and transferred to PVDF membranes (Bio-Rad, 1620233) using the Trans-Blot® Turbo^™ Transfer Pack and System (Bio-Rad). Membranes were blocked with TBST (Cell Signaling Technology, 9997S) containing 5% skim milk for 1 h and incubated overnight at 4°C with various primary antibodies. Following 3 washes in TBST, membranes were incubated with goat anti-rabbit or anti-mouse IgG HRP secondary antibody (Cell Signaling Technology, 7074 or 7076) at room temperature for 1 h and washed. Chemiluminescence substrate was applied using SuperSignal^™ West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, 34080) or SuperSignal^™ West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, 34095) and blots were analyzed using the ChemiDoc^™ Touch Imaging System (Bio-Rad).

Cells were lysed with 1× cell lysis buffer (Cell Signaling Technology, 9803) containing protease inhibitor (Sigma-Aldrich, P8340) on ice for 10 min, homogenized by passing through a 21-gauge needle, and centrifuged at 14,000 g for 15 min at 4°C to pellet the cell debris. Protein was quantified using the BCA assay (Thermo Fisher Scientific, 23225) and 20 μg of each sample was resolved on SDS-PAGE and transferred to nitrocellulose membrane. Membranes were blocked with TBST containing 5% skim milk for 1 h and incubated overnight at 4°C with various primary antibodies followed by incubation with goat anti-rabbit or anti-mouse IgG HRP secondary antibody (Cell Signaling Technology, 7074 or 7076) at room temperature for 1 hr Chemiluminescence substrate was applied using SuperSignal™ West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, 34080) and blots were analyzed using the ChemiDoc™ Touch Imaging System (Bio-Rad). Semi-quantification of data was performed using Image Lab (Bio-Rad).