Knockdown of Myo9b causes mislocalization and diminished activation of Src kinase.

<p><i>A</i>, Immunocytochemical labeling of podosome belts in mature osteoclasts demonstrates that Src is mislocalized when Myo9b levels are suppressed. Arrows indicate the normal “railroad track” distribution of Src around podosome belts in control cells, while Src is present in the F-actin core of podosomes in siRNA-treated cells. <i>B</i>, Immunoprecipitation of total cellular Src kinase followed by Western blot with a phospho-tyrosine antibody demonstrates that siRNA treatment diminishes Src phosphorylation without affecting Src protein levels. One lane of immunoprecipitation with the mouse IgG MOPC-21 was added as a negative control. <i>C</i>, Immunocytochemical labeling of osteoclasts with an antibody against phosphorylated Src shows its relative absence in siRNA-treated cells. <i>D</i>, Inhibition of Src kinase causes loss of podosome belts in untreated and control osteoclasts, but does not further diminish podosome belts in cells with suppressed levels of Myo9b. <i>E</i>, Immunocytochemical analysis shows that Pyk2 distribution does not change with siRNA treatment. For all photomicrographs in this figure, scale bars = 20 µm.