Criterion tgx stain free 4 15 gradient gels

Cells were lysed in SDS lysis buffer (4 % SDS, 20 % glycerol, 125 mM Tris-HCl pH = 6.8) supplemented with protease inhibitors (cOmplete^TM Mini (Roche)). The insoluble material was removed by centrifugation at 15,000 g for 15 min. Protein concentrations were determined by BioRad™ DC-Protein Assay using bovine serum albumin (BSA) as standard. Following the normalization of protein concentrations, the lysates were mixed with an equal volume of 2X Laemmli sample buffer and incubated for 5 min at 95 °C before separation by SDS-PAGE on Criterion TGX stain-free 4-15 % gradient gels (BioRad). Following SDS-PAGE, the proteins were transferred to polyvinylidene fluoride membranes (30V overnight at 4 °C). The membranes were then blocked with BSA (Sigma-Aldrich) dissolved in PBS/Tween-20 (3 % BSA, 0.5 % Tween-20 for 1-2 hours), followed by immunoblotting with the primary antibody: rabbit anti-HIF-3α (Sigma-Aldrich, AV39936 (1:800)); rabbit anti-β-Actin (1:1000, ab1801; Abcam). The rabbit anti-HIF-3α antibody only recognizes the α1, α2 and α3 isoforms of HIF-3α. After the washing steps, the membranes were incubated with goat anti-rabbit IgG (H+L chains) or HRP-conjugated secondary antibodies (BioRad) and detected using SuperSignal West Pico ECL (Thermo Fisher Scientific). Densitometry was performed using Image Lab software v. 4.1 (BioRad).

Cells were lysed in SDS lysis buffer (4% SDS, 20% glycerol, 125 mM Tris–HCl pH = 6.8) supplemented with protease inhibitors (cOmplete ™ Mini (Roche)). The insoluble material was removed by centrifugation at 15,000g for 15 min. Protein concentrations were determined by Bio-Rad™ DC-Protein Assay using bovine serum albumin (BSA) as standard. Following the normalization of protein concentrations, the lysates were mixed with an equal volume of 6X Laemmli sample buffer and incubated for 5 min at 95 °C prior to separation by SDS PAGE on Criterion TGX stain-free 4–15% gradient gels (Bio-Rad). Following SDS-PAGE, the proteins were transferred to polyvinylidene fluoride membranes (300 mA 4 h at 4 °C). The membranes were then blocked with BSA (Sigma-Aldrich) dissolved in PBS/Tween-20 (3% BSA, 0.5% Tween-20 for 1–2 h), followed by immunoblotting with the primary antibody: mouse anti-HIF-1α (1:2000, ab16066; Abcam); rabbit anti-HIF-2α (1:1000, ab199, Abcam); rabbit anti-β-Actin (1:1000, ab1801; Abcam). After the washing steps, the membranes were incubated with goat anti-rabbit IgG (H + L chains) or with goat anti-mouse IgG (H + L) HRP-conjugated secondary antibodies (Bio-Rad) and detected using SuperSignal West Pico ECL (Thermo Fisher Scientific). Densitometry was performed using Image Lab software v. 4.1 (Bio-Rad).