Anti c myc antibody 9e10

The antibodies used were as follows: anti-c-Myc antibody (9E10, 1:1000 for IB analysis, Santa Cruz), anti-Flag antibody (F1804, 1:1000 for IB analysis, Sigma), anti-HA antibody (1:5000 for IB analysis, Covance), anti-GAPDH antibody (SC-47724, 1:1000 for IB analysis, Santa Cruz), anti-α-tubulin antibody (EPR1333, 1:10 000 for IB analysis, Epitomics), anti-HIF-2α antibody (AB199, 1:1000 for IB analysis, Abcam), anti-HIF-1α antibody (A6265, 1:1000 for IB analysis, Abclone), anti-HIF-OH antibody (3434S, 1:1000 for IB analysis, Cell Signaling).
Western blot was performed as described previously (31 (link)). The Fuji Film LAS4000 mini luminescent image analyzer was used for photographing the blots. Multi Gauge V3.0 was used for quantifying the protein levels based on the band density obtained in western blot analysis.

pEF6-CSPG4-myc-his plasmid was purchased from Addgene (Cambridge, MA). DNA was ligated into pcDNA™3.1⁽⁺⁾ (Thermo Fisher Scientific), amplified, and sequence confirmed. Human embryonic kidney 293T cells (ATCC) were cotransfected with human-V5-ABCA8-pcDNA3.1 and myc-CSPG4 using Lipofectamine 300 reagent (Thermo Fisher Scientific). Twenty-four hours after transfection, cells were lysed in 10 mM Tris-HCl, 0.1% Triton, 150 mM NaCl, pH 8.0, 1× protease inhibitor cocktail (Roche, Basel, Switzerland), and centrifuged at 14,000 g for 20 min at 4°C. Protein A sepharose beads (50 μl; GE Healthcare, MA) were incubated for 2 h at 4°C with 2 μg anti-V5 antibody (Invitrogen) or anti-c-myc antibody 9E10 (Santa Cruz Biotechnology, Dallas, TX). The cell lysates (500 μg) were incubated with the beads overnight at 4°C. Nonspecific binding was assessed using beads incubated without protein lysate. Beads were washed in cold homogenization buffer containing 0.4% Tween-20, and the bound proteins were detached using sample buffer containing 200 mM dithiothreitol at 100°C for 5 min. Samples were analyzed by Western blotting to detect human ABCA8 (anti-V5 [Invitrogen]) and anti-c-myc antibody (9E10) to detect tagged CSPG4.