Fig. 4.

Overexpression of a mutant RhoGAP22 that cannot bind 14-3-3 results in decreased random cell motility. (A) NIH 3T3 fibroblasts were infected with pMIG retrovirus encoding empty vector (pMIG), FLAG-RhoGAP22 (WT), or mutants of FLAG-RhoGAP22. The pMIG retrovirus also independently expresses eGFP using an IRES. Cells were subjected to FACS for the median 20% of GFP-expressing cells of each line. Cells from each line were lysed, and lysates were immunoblotted with the indicated antibodies. (B) NIH 3T3 fibroblasts expressing various RhoGAP22 constructs (green) or vector only (red) were imaged every 10 min for 14 h. Tracks depict migration of individual cells. (C) Cell displacement over 14 h was calculated using Imaris software and reported as the mean ratio of RhoGAP22 mutant-expressing cell displacement to the displacement of cells expressing empty vector. Values are means from three experiments; error bars show standard deviations. Significance was determined by one-way repeated-measures ANOVA. *, P < 0.05.