Figure 4.

Inhibition of thrombin-induced Rho activation by expression of Gα13-ct and p115RGS in RA synovial fibroblasts. Rheumatoid arthritis (RA) synovial fibroblasts (SFs) were or were not infected with adenoviruses encoding green-fluorescent protein (GFP) (control vector), the C-terminal regions of Gα13 (Gα13-ct), or P115RGS. Cells that were not infected with adenovirus were incubated in the medium alone. Cells were then cultured for 48 hours in DMEM containing 1% FCS, then stimulated with 10 units/ml thrombin for 1 minute or were loaded with GTPγS (positive control), after which they were lysed to measure Rho activity. Rho activity is indicated by the amount of Rho bound by the Rhotekin-Rho-binding domain (RBD) (top). The percentage of activated Rho (graph) is expressed as a ratio relative to 4% of total Rho (4% of total protein used in the RBD bead pull-down experiments). Results are representative of three experiments. Western blot analysis confirmed that equal amounts of total Rho were used for the pull-down assay under each condition (data not shown). (-), cells without infection; p115RGS, regulator of G-protein signaling domain of p115Rho guanine nucleotide exchange factor.