Nischarin interacts with PAK1/2 and regulates PAK activity.

<p>Lysates (500 μl, concentration 3 μg/μl) of rat cortical neurons were incubated with an anti-rabbit PAK1 polyclonal antibody, an anti-rabbit PAK2 polyclonal antibody or an anti-mouse Nischarin monoclonal antibody and their IgG then precipitated with G-Sepharose protein beads. <b>(A)</b> The Nischarin immunoprecipitates were blotted with anti-PAK1 antibody (top panel) or with anti-PAK2 antibody (bottom panel). <b>(B)</b> PAK1 or PAK2 immunoprecipitates were blotted with anti-Nischarin antibody. IgG was used as the negative control and 5% cell lysates (input) was added in the gel in each experiment to be a positive control. IP EX: immunoprecipitation extract. <b>(C)</b> Immunofluorescence staining was performed on primary cultured hippocampal neurons (10 DIV). Strong staining for Nischarin (green) co-localized both with PAK1 (red, left panel) and PAK2 (red, right panel) in the perinuclear region and the dendrites (arrows). Scale bars: 10 μm. <b>(D, E)</b> Nischarin-shRNA effectively suppresses the expression of Nischarin protein and stimulates the phosphorylation of both PAK1 and PAK2, in Neuro-2a cells (n = 3). Asterisks indicate significant differences between ctl-shRNA and Nis-shRNA (*<i>p</i><0.05, ***<i>p</i><0.001, n = 3). Data presented are the mean ± SEM.