HIF-1α is functionally active in CLL B cells. (A) HIF-1α accumulates in the nuclei of CLL B cells. Nuclear extracts prepared from freshly isolated CLL B cells (n = 8) were analyzed for nuclear accumulation of HIF-1α by Western blot. An antibody to ORC2 was used for the purity of nuclear preparation. Analysis of the cytoplasmic fractions for HIF-1α was also performed by Western blot. An antibody to Cu/Zn SOD was used to indicate the purity of the cytoplasmic preparations. (B) HIF-1α forms a complex with p300 and P-STAT3. Freshly isolated CLL B-cell lysates (P4-6) were used to immunoprecipitate HIF-1α or p300 using specific mouse monoclonal antibodies. Mouse whole immunoglobulin (IgG) was used as control antibody for immunoprecipitation. The immunoprecipitated complexes were subjected to Western blot analyses using specific antibodies to p300, phospho-STAT3, or HIF-1α, as indicated. Endogenous expression levels of HIF-1α, p300, and phospho-STAT3 in the CLL B-cell lysates used in these experiments are shown by Western blot analyses using specific antibodies. Actin was used as the loading control. (C) HIF-1α and P-STAT3 form an active complex at the VEGF promoter in CLL B cells. Chromatin immunoprecipitation assay was performed with an antibody to HIF-1α, phospho-STAT3, or phospho-RNA polymerase II or control mouse IgG using the cross-linked nuclear extracts from CLL B cells (P5, P6, and P23) as described in “ChIP assay.” The immunoprecipitated DNA was purified and the region from −1386 to −1036 bp of the human VEGF promoter was amplified by PCR. Input DNA is indicated.