Fig. 2

HCR co-localizes with astrin at the centrosome and mitotic spindle. A HeLa cells stably expressing HCR-GFP (green) were stained with an astrin antibody (red) and DAPI (blue) followed by confocal microscopy analysis (left panel); HeLa cells transfected with GFP-astrin (green) were stained with HCR (red) and gamma-tubulin (cyan) antibodies and DAPI (blue) for nuclear staining (right panel); scale bars, 10 μm. B Mitotic HeLa cells stably transfected with HCR-GFP (green) were stained with astrin (red), gamma-tubulin (cyan), and DAPI (blue); scale bars, 10 μm. C HeLa cell lysates were immunoprecipitated with control rabbit IgG or anti-HCR and detected by immunoblotting for HCR and PCM1. Beta-actin was used as a negative control (left panel) or immunoprecipitated with astrin and analyzed by western blotting for astrin and PCM1. Beta-actin was used as negative control (right panel). D HeLa cells were synchronized and stained with PCM1 (green), HCR (red), and DAPI (blue); scale bars, 10 μm; inset scale bars, 1 μm. E HeLa cells were treated with DMSO, 2 μg/ml nocodazole, or 1 μM paclitaxel and then stained with anti-HCR (red), anti-gamma-tubulin (green), and DAPI (blue); scale bars, 10 μm. F HeLa cells were transfected with the indicated siRNA and then immunostained for HCR (red) and gamma-tubulin (green). The nucleus was stained with DAPI (blue); scale bars, 10 μm; inset scale bars, 1 μm