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Fig. 4. (A-F) NIH-3T3 cells were transfected with vector EGFP, and EGFP-tagged GEF-H1 alleles: GEF-H1S, GEF-H1S-S810A, GEF-H1-Q312M,R313G, GEF-H1S-S67A and GEF-H1S-S67,810A. GFP fluorescence was used to detect expression of GEF-H1 (left panels) and fluorescent-phallicidin was used to monitor cytoskeletal rearrangements (right panels). Localization of GEF-H1S proteins in NIH-3T3 fibroblasts was detected by expressing as EGFP fusion proteins and analyzing by fluorescence microscopy. (G-L) Overlapping F-actin localization. (M) Regulation of Rho activity by GEF-H1 proteins was measured by precipitation of Rho-GTP with GST-RBD from lysates co-expressing GEF-H1 and Rho. The amount of RhoA bound to RBD and the level of RhoA expression in whole cells lysates were analyzed by western blotting and detected with anti-Myc antibody. GEF-H1 was detected with anti-HA antibody. (N) Histogram displaying fold activation of Rho. Measurements were by densitometry using the western blots of two individual experiments.





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