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Figure 4. Agrin-dependent enhancement of cortactin tyrosine phosphorylation.Cultured C2 mouse myotubes were exposed to medium without (-) or with added agrin (+) before preparing extracts for immuno-precipitation (A) with a monoclonal antibody against cortactin (IP: cort) or an unrelated protein (IP: ctl). When these samples were immuno-blotted for cortactin (IB: cort) and total phosphotyrosine (IB: PY; mAb4G10), cortactin was found to be captured only by the anti-cortactin antibody (upper lanes), and anti-phosphotyrosine staining showed that cortactin from extracts of agrin-treated cells was tyrosine phosphorylated significantly more than that captured from control extracts (lower lanes). This increase in cortactin phosphorylation was quantified from four experiments (A, graph) by measuring band densities, normalizing for cortactin loading (see Methods), and calculating the phosphotyrosine level change relative to control. B. To test whether the src-target sites in cortactin were phosphorylated in response to agrin-treatment, myotube extracts were blotted with antibodies against total cortactin and cortactin phosphorylated on Y421. Agrin-treatment did not alter the amount of cortactin present in extracts (upper lanes) but the staining of cortactin by the anti-Y421-phospho-cortactin antibody (IB: pCort) was enhanced by agrin-treatment more than two-fold, as shown by quantification from three experiments (B, graph). Positions of pre-stained MW markers (Bio-Rad) are indicated on the right side of blots, and in the graphs * represents P<0.02 in t-tests. Image published in: Madhavan R et al. (2009) Madhavan et al. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license Larger Image Printer Friendly View

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