2.9. IKZF1, IKZF3 and ABC50 Immunoblotting

Cells were lysed in TBS buffer containing 1% Triton X-100 and Complete® protease inhibitor cocktail (Roche) for 15 min on ice with occasional vortexing. Insoluble material was centrifuged (12,000 rpm, 15 min, 4 °C) and supernatants were collected. Total protein concentration was measured with DC Protein Assay (Bio-Rad, Hercules, CA, USA). Cell lysates containing 30 µg of total protein were mixed with Laemli buffer supplemented with SDS and β-mercaptoethanol, denatured (10 min, 95 °C) and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis (4% stacking and 10% resolving gels). Resolved proteins were electrotransferred (semi-dry, 90 min, 1.5 mA/cm²) to a 0.45 µm nitrocellulose membrane (Thermo Scientific, Waltham, MA, USA). Membranes were blocked for 2 hours in TBST buffer (20 mM Tris-HCl pH 7.5, 0.9% NaCl, 0.1% Tween 20) containing 5% BSA (BioShop, Burlington, ON, Canada) and probed with primary antibodies (overnight at 4 °C) diluted in TBST buffer with 0.5% BSA. Tubulin served as a loading control for IB experiments. Anti IKZF3 (rabbit monoclonal, dilution 1:500), anti IKZF1 (rabbit polyclonal, dilution 1:500) were from Abcam. Anti IRF4 (rabbit polyclonal, dilution 1:500) were from Cell Signaling Technology. Anti α-tubulin (mouse monoclonal IgG, 1:1000) was from Sigma Aldrich. Anti ABC50 antibodies (mouse monoclonal ABC5H06, dilution 1:100) was from Abcam. After extensive washing with TBST buffer, membranes were probed with secondary goat anti-rabbit or goat anti-mouse HRP-conjugated IgG (1:5000 in TBST, Santa Cruz Biotech, Dallas, TX, USA) for 45 min at room temperature. Membranes were washed in TBST buffer and the chemiluminescent signal was developed with ECL Plus Western Blotting Substrate (Thermo Scientific) and visualized in Syngene G:Box detection system. Protein bands intensities were analysed using Image Quant (version 5.0, GE Healthcare Life Sciences, Pittsburgh, PA, USA).