When indicated, NB4 cells (shCTRL or shHIFs) were treated for 24 h with 100 μM CoCl2 before lysis. For Western blot, proteins were extracted with RIPA buffer (Sigma) supplemented with protease inhibitor cocktail (Roche); for luciferase assays, cells were lyzed in passive lysis buffer (Promega); for co-immunoprecipitation experiments, cells were lyzed after mild cross-linking with 0.4% formaldehyde (7 min at RT) in CO-IP buffer (10 mM NaCl, 10 mM Tris–HCl pH 7.5, 5 mM MgCl2, 0.5% CHAPS) supplemented with protease and phosphatase inhibitors (Pierce) and then briefly sonicated to extract nuclear proteins. Total lysates were immunoprecipitated with an antibody against PML (PG-M3; Santa Cruz).
Cell extracts and immunoprecipitates were resolved by SDS–PAGE 7.5–10% and transferred to a PVDF membrane (Biorad). Non-specific binding was blocked in 5% non-fat milk for 1 h at RT and blotted with the following antibodies: rabbit polyclonal HIF-1α (Cayman), rabbit polyclonal PML (Santa Cruz) and rabbit polyclonal PML (Novus). Mouse β-actin (Sigma) was used as internal loading control.
Cell extracts and immunoprecipitates were resolved by SDS–PAGE 7.5–10% and transferred to a PVDF membrane (Biorad). Non-specific binding was blocked in 5% non-fat milk for 1 h at RT and blotted with the following antibodies: rabbit polyclonal HIF-1α (Cayman), rabbit polyclonal PML (Santa Cruz) and rabbit polyclonal PML (Novus). Mouse β-actin (Sigma) was used as internal loading control.