Nucleolin anchors to intracellular MyH9 during angiogenic. (A) Cell-free lysate was prepared from HMECs grown on immobilized FN or PLL. Co-IP was performed with antinucleolin Ab and purified rabbit IgG as a control. The arrows indicate the precipitated protein band, which was identified as MyH9 by MALDI-TOF analysis. The SDS-PAGE gel was silver stained and the finger printing map was indicated. (B) Co-IP was performed as described for panel A, and the precipitated proteins were then blotted (IB) with antibodies against MyH9 or nucleolin. The nuclei of HMECs grown on immobilized FN were extracted; then both nuclear and extranuclear fraction were lysed to perform co-IP with the same condition. (C) Distribution of actin filament (green) and MyH9 (red) in HMECs grown on immobilized FN or PLL were analyzed. Bar, 10 μm. (D) Upon attachment to matrix and stimulation by growth factors, endothelial cells form a tubule structure; meanwhile, nucleolin (red) translocated to extra-nuclei and colocalized with MyH9 (green). Nucleolin and MyH9 were stained by indirect immunofluorescence. Bar, 10 μm. (E) Under normal conditions without matrix and growth factors supplements, nucleolin (green) stayed in nuclei and MyH9 (red) was located in cytosol. The nuclei (blue) were counterstained with DAPI. Bar, 10 μm. (F) The MyH9ΔN-GFP was pulled down by antinucleolin Ab.