FIGURE 6.

pVHL mediates degradation of HIF-1α(P402A/P563A). A, Co(II) does not upregulate HIF-1α protein levels in a nonfunctional pVHL-expressing cell line. SKRC-17 (not expressing HIF-1α) and SKRC-7 (expressing HIF-1α) cell lines were treated with CoCl₂ for 8 h, and whole cell extracts were prepared. Endogenous HIF-1α protein levels were analyzed (150 μg of extracts) by immunoblotting using anti-HIF-1α (α-HIF-1α), or anti-actin (α-Act) antibodies. As a positive control, 25 μg of extracts from HEK 293A (A) cells transfected with pFLAG-mHIF-1α and treated with CoCl₂ were used. B, knocking-down pVHL expression by RNA interference leads to accumulation of HIF-1α(P402A/P563A). Stable cells expressing wild-type (wt 4) or the mHIF-1α double proline mutant (PP-A 9) were transfected with negative control (C) or VHL (V) siRNA. B, upper panel. Whole cell extracts were separated by SDS-PAGE and analyzed by immunoblotting using anti-FLAG (α-FLAG), anti-VHL (α-VHL), or anti-actin (α-Actin) antibodies. Cells were treated with CoCl₂ as indicated (+). B, lower panel, RNA level analysis following siRNA experiments targeting VHL expression. Semiquantitative RT-PCR was performed using primers to FLAG-HIF-1α, VHL, and actin. C, pVHL mediates HIF-1α(P402A/P563A) ubiquitination. Stable cells expressing p1 (puromycin control), wild-type HIF-1α (wt 4), or HIF-1α mutant (PP-A 9) were transfected with siRNA as in B, and exposed to MG132. C, upper panel, whole cell extracts (Inputs) and immunoprecipitated FLAG-tagged proteins (IP) were analyzed using anti-FLAG (α-FLAG), anti-ubiquitin (α-Ub), or anti-actin (α-Actin) antibodies. C, lower panel, analysis of VHL mRNA levels following RNAi experiments. Semiquantitative RT-PCR was performed using primers to VHL and actin.