FIGURE 3.

The dynein motor protein mediates the microtubule association of HIF-1α. A, PC3 cells were exposed to 4 h hypoxia and subjected to immunoprecipitation using an antibody against the intermediate chain of dynein. Immunoprecipitates and 50 μg of whole cell extract (WCE) were resolved by SDS-PAGE and immunoblotted with antibodies against HIF-1α or α-tubulin. B, PC3 cells were transiently transfected with pCMVH50 dynamitin vector (encoding a c-Myc-tagged human dynamitin), or the unrelated pCMVβ-galactosidase vector as a control, and subjected to 6 h hypoxia. Cells were fixed and processed for double immunofluorescence labeling using antibodies against HIF-1α (green) or c-Myc (red). Solid arrows point to cells overexpressing dynamitin. Arrowheads point to non-transfected cells. Scale bars: 5 μm. C, importin-α co-precipitates with tubulin. PC3 cells stably expressing mCherry-tubulin were subjected to immunoprecipitation using an antibody against rat α-tubulin (YL1/2) or matching rat IgG. Immunoprecipitates were resolved on SDS-PAGE along with 50 μg of WCE and immunoblotted with an antibody against importin-α1 (karyopherin α2). 1 μg of the antibody used for the IP was also loaded as a control for the heavy chains (Ab).