Figure 7.

Analysis of HIF-1α ubiquitination. (A) 293 cells were cotransfected with expression vectors encoding HIF-1α and either no protein (lanes 1–2) or His₆-ubiquitin (lanes 3–4). Cells were exposed to 20% or 1% O₂ for 4 h, and lysates were prepared. Two hundred-microgram aliquots of total protein were incubated with nickel resin and washed with buffer containing 75 mM imidazole. Aliquots of unpurified (Top) and nickel resin-purified (Bottom) lysates were subjected to SDS/PAGE and immunoblot assay by using an anti-HIF-1α polyclonal Ab. (B) Levels of total and ubiquitinated HIF-1α in lanes 3–4 were quantified by densitometry. The ratio of ubiquitinated/total HIF-1α was determined and normalized to the result obtained for the lysates analyzed in lane 4 to yield the relative HIF-1α ubiquitination. (C) Cells were cotransfected with: pSVβgal; expression vector encoding HIF-1α(1–391/512–826) that was either wild type (WT; lanes 1–3) or contained the S551G/T552A missense mutations (M; lane 4); and vector encoding either no protein (lanes 1–2) or His₆-ubiquitin (lanes 3–4). Cells were exposed to 20% or 1% O₂ for 4 h, and lysates were prepared. Aliquots of total protein (normalized to β-gal) were incubated with nickel resin and washed with RIPA buffer (0.15 mM NaCl/0.05 mM Tris⋅HCl, pH 7.2/1% Triton X-100/1% sodium deoxycholate/0.1% SDS) containing 75 mM imidazole. Aliquots of unpurified (Top) or resin-purified (Bottom) lysates were subjected to immunoblot assay by using an anti-HIF-1α polyclonal Ab. (D) Levels of total and ubiquitinated wild-type and mutant HIF-1α (1–391/512–826) in lanes 3–4 were quantified by densitometry. The ratio of ubiquitinated/total HIF-1α was determined and normalized to the result obtained for the lysate analyzed in lane 4 to yield the relative HIF-1α ubiquitination.