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.