Anti β actin clone ac 15

One million cells each were lysed in pre-cooled RIPA buffer (Pierce, Thermo Fisher Scientific) containing phosphatase and proteinase inhibitors and 2.5 mmol/l dithiothreitol (Sigma-Aldrich). Equal amounts of proteins (15 µg) were loaded on a 10% polyacrylamide gel (SDS-PAGE), electrophoresed and then blotted by semi-dry transfer onto a nitrocellulose membrane (Schleicher & Schuell). After a blocking step with 5% non-fat milk (Merck KGaA) for 1 h, the membranes were incubated with the indicated antibodies overnight at 4 °C. After washing with PBS, membranes were incubated with 1:10,000 diluted horseradish peroxidase­conjugated goat anti-rabbit or rabbit anti-mouse (both antibodies from Dako, Denmark) for 60 min at room temperature. The peroxidase-conjugated monoclonal mouse antibodies anti-GAPDH (clone GAPDH-71.1) and anti-β-actin (clone AC-15) were used as loading control (at 1:10,000 dilution, Sigma-Aldrich). lmmunoblots were visualized by highly sensitive chemiluminescent detection reagent (Amersham, GE Healthcare, UK). All primary antibodies were used at a 1:1,000 dilution in TBST (5 mM TRIS, 15 mM NaCl, 0.1% Tween 20, pH 7.5) with 5% non-fat milk (Merck KGaA, Germany) for western blots. Anti-BRD4 (E2A7X), anti-cereblon (D8H3S), and anti-VHL (#68547) were purchased from Cell Signaling Technology and anti-MCY (Y69) were purchased from Abcam.