FIG. 6.

Activation of small G proteins by FA-CRKL. (A) Immunoblot assay for expression levels of FA-CRKL compared to those of endogenous CRKL in NIH 3T3 cells expressing empty vector (CTR; lane 1), FA-CRKL, or FA-CRKL(ΔSH3n). (B) Pulldown assays for GTP-loaded Rac1, Cdc42, and Ras. Overall levels of these small G proteins were determined in parallel by using total cell lysates from NIH 3T3 cells expressing empty vector (CTR; lane 1), FA-CRKL (lane 2), or FA-CRKL(ΔSH3n) (lane 3) (shown at left). GTP-loaded forms were pulled down as described in Materials and Methods, and their levels were determined by immunoblotting by using specific antibodies to these small G proteins (shown at right). Levels of the GTP-loaded form were compared to total levels (not discriminating among GTP- and GDP-bound forms) of the G protein determined in total cell lysates. Experiments were repeated three times and yielded similar results. (C) The effects of dominant-negative forms of small G proteins were evaluated by counting the number of cells that show multiple membrane protrusions induced by FA-CRKL. Increasing doses of expression plasmids for Rac1 N17, Cdc42 N17, RhoA N17, or Ras N17 were cotransfected into NIH 3T3 cells coexpressing FA-CRKL at a fixed dose of 0.1 μg/well. Cells were stained with phalloidin to aid scoring samples. Values indicate the percentage of cells with lamellipodia among the total number of GFP-positive transfected cells. Error bars indicate the standard deviation of the sample values obtained in three independent sets of samples. (D) Subcellular localization of Dock1 in NIH 3T3 cells that express either FA-CRKL or FA-CRKL(ΔSH3n). Localization of fusion proteins was determined by GFP fluorescence. A part of the FA-CRKL-expressing cell highlighted by the white square was enlarged to show details of colocalization of FA-CRKL with Dock1.