Fig. 1. Stable FRNK expression in v-Src3T3s does not inhibit cell motility. (A) Equal protein lysates of either NIH-3T3 or v-Src3T3 fibroblasts were analyzed by blotting with a v-Src-specific mAb (top panel) and re-probed with an HA tag mAb to visualize FRNK (clones R2, A2 and O4) or FRNK S-1034 expression (lower panel). (B) c-Src and v-Src were immunoprecipitated (mAb 2-17) from lysates of serum-starved cells and were analyzed for associated in vitro kinase (IVK) activity using GST–FAK-CT as a substrate. (C) Haptotaxis motility was assayed with immobilized FN and the indicated cells. Random cell motility was measured in the presence of BSA. Values are means ± SD from four experiments. (D) Chemotaxis motility was assayed on collagen-coated membranes with the indicated cells using a serum stimulus added to the lower chamber. Random cell motility was measured in the presence of BSA. Values are means ± SD from three experiments. (E) Wound-healing scratch motility assays were performed with the indicated cells plated onto Matrigel-coated glass slides in the presence of serum. Cell migration was assessed by 1 mm grid comparisons of four image sets at 0 and 24 h; representative images are shown.