Mini tgx gel

Proteins from cultured MTFs were extracted using ice-cold RIPA buffer (Thermo Fisher Scientific, MA, USA) containing protease inhibitors by vortexing for 30 min. The protein samples were quantified while using the BCA Protein Assay kit (Pierce Biotechnology, Rockford, IL, USA; #23 227). Samples containing equal concentrations of protein were loaded into wells containing 4–20% mini-TGX gels (Bio-Rad, Hercules, CA, USA; #456–1096), and electrophoresis was performed. Subsequently, the samples were transferred onto polyvinylidene difluoride membranes. The blots were probed overnight at 4 °C with a primary antibody diluted in TBST and 1% BSA. Table 1 lists the primary antibodies used in the study. Following three washes in TBST (30 min. each), the blots were incubated for 1 h with 1:2000 dilutions of anti-mouse and anti-rabbit IgG and HRP-linked antibodies (Santa Cruz Biotechnology, 1:1000) prepared in TBST and 1% BSA. The ECL Western Blotting Substrate (Thermo Scientific, Rockford, IL, USA; #32106) and HyBlot CL autoradiography film (Denville Scientific, Metuchen, NJ, USA; #E3018) were used for signal testing. β-actin was used as the control for normalization.

Whole cell lysates (40–80 μg protein in RIPA buffer with Complete, Roche, Switzerland) were electrophoretically separated (7.5% Mini TGX gel, BioRad Laboratories CA). Protein detection was performed using anti-HIF1α (Becton Dickinson, NJ), anti-HIF1β (Ab126985) anti-HIF2α (Ab199, Abcam, UK), anti-EGFR (DAK-H1-WT, Dako, Denmark), ERα (Cell Signaling Technologies, MA), actin (MP Biomedicals, CA) and SDHA (Ab14715, Abcam).

Whole cell lysates (40–80 μg protein in RIPA buffer with Complete, Roche, Switzerland) were electrophoretically separated (7.5% Mini TGX gel, BioRad Laboratories CA, according to manufacturers instructions). Protein detection was performed using antibodies against HIF-1α (Becton Dickinson, NJ), ERα (Cell Signaling Technologies, MA), actin (MP Biomedicals, CA) and SDHA (Ab14715, Abcam).

Whole cell lysates were made from pelleting 1 × 10⁷ cells at 4C at 3000 x g and resuspending in lysis buffer (20 mM Tris-HCl, pH 8.0; 150 mM KCl; 5 mM MgCl2; 250 mM Sucrose; 1 mM DTT; 10 mM Digitonin; 1 mg/ml Sodium Heparin; 1 mM Pefabloc SC; 0.5 U/μl DNaseI; 1 U/μl SUPERaseIN). Cells were incubated in lysis buffer for 10 min on ice and passed through a 30G needle 5x. Insoluble material was pelleted at 6000 x g for 10 min at 4C. Lysate (1 × 10⁶ cells/sample) was run on a 4–20% TGX mini-gel (Bio-Rad) for 45 min at 200 V and transferred onto 0.2 μm nitrocellulose membrane using the Trans-Blot Turbo Transfer System (Bio-Rad) with semi-dry settings 25V for min. The blot was blocked for 30 min with Odyssey PBS Block (Li-cor). The blot was probed with biotinylated jacalin (1:4,000; Vector Labs) and E7 anti-tubulin antibody (1:10,000; Developmental Studies Hybridoma Bank) diluted in block for 1 hr, and then with IRDye 800 streptavidin (1:1,000; Li-cor) and IRDye 700 mouse (1:1,000; Li-cor) in PBST [PBS with %1 Tween 20 (v/v)]. Blot was imaged on Licor Odyssey (Figure 4—figure supplement 1).

Protein (and crude viral lysates) were mixed at an equal ratio with a 2X reducing sample SDS-PAGE buffer, heated to 75 °C for 20 minutes, separated on a 4–20% precast TGX mini-gel (Biorad), and transferred onto a PVDF membrane (Millipore). Membranes were blocked with 5% BSA in TBS with 0.05% Tween (TBST) for 30 minutes, before incubating at 4 °C overnight with agitation with the appropriate antibody diluted in 5% BSA in TBST. Following three washes in TBST for 10 minutes each, membranes were incubated at room temperature with mild agitation with an appropriate secondary HRP antibody (Sigma Aldrich, 1:5000) diluted in 5% BSA in TBST, washed three times with TBST for 10 minutes each, and developed with enhanced chemiluminescent substrate (Amersham-Pharmacia Biotech, USA).