Fig. 2.

FER phosphorylates CRMP2 at Y479 and Y499. a, b Recombinant GST-CRMP2 (residues 1–516), GST-CRMP2-Y479F (residues 1–516), or GST-CRMP2-Y499F (residues 1–516) were purified and used for in vitro kinase assays in the absence or presence of active GST-tagged truncated FER protein (residues 521–822). The samples were analyzed by SDS-PAGE, and immunoblotted using the indicated antibodies. IB immunoblot. c, d Lysates of the indicated ovarian cancer cell lines were immunoprecipitated using an anti-CRMP2 antibody. The samples were analyzed by SDS-PAGE, and immunoblotted with the indicated antibodies. CRMP2 was loaded as a control for protein input. e Lysates of the ovarian cancer cell line OVCA432, which was treated with non-targeting (nt) siRNA or FER siRNA, were immunoprecipitated with anti-CRMP2 antibody. The samples were analyzed by SDS-PAGE, and immunoblotted with the indicated antibodies. f, g The indicated recombinant CRMP2 wild-type or mutant proteins were purified and incubated with paclitaxel-stabilized rhodamine-labeled microtubules for 40 min at room temperature. Bar plots represent the mean + s.e.m. of microtubule width from at least 150 individual microtubules per condition tested. Shown are typical results from at least three independent replicates. Scale bar is 10 µm