Pax3-FOXO1 is phosphorylated on Ser205 by CK2. Bacterially expressed and purified GST (27kD), GST-Pax3 (74kD), or GST-Pax3-FOXO1 (125kD) were phosphorylated in vitro using purified CK2 (A and B) or proliferating primary myoblast total cell extracts (C and D) as described in the Experimental procedures. The phosphorylated proteins were eluted from the resin, separated by 8% SDS-PAGE, and visualized by Coomasie staining (A and C, left panels), autoradiography (A and C, middle and right panels) or Western blot analysis using our phospho-specific anti-Pax3(p205) antibody (12) (B and D). The arrows in panels A and C indicate the major protein species for GST (lower arrow), GST-Pax3 (middle arrow) and GST-Pax3-FOXO1 (upper arrow). The arrows in panels B and D indicate the major protein species for GST-Pax3 (lower arrow) and GST-Pax3-FOXO1 (upper arrow). All other observable protein species are degradation products, which are commonly seen in these protein preparations.