Fig. 3
Inhibitory effects of RGS2 and RGS4 on PAR4/Gαq- and PAR4/Gα12/13-mediated signaling. a 293 T cells were transfected with PAR4 (1.0 μg) together with RGS2-HA (1.0 μg), RGS4-HA (1.0 μg) or RGS16-HA (1 μg), as indicated. b HT29 cells were transfected with RGS2-HA (1.0 μg), RGS4-HA (1.0 μg) or RGS16-HA (1.0 μg). a–b After transfection, cells were stimulated with 20 μM AYPGKF for 7 min and immunoblotting performed on cell lysates using antibodies against p-ERK and total ERK. Expression of RGS proteins was confirmed using antibodies against HA. Densitometry analysis was conducted to determine the relative band intensity of p-ERK/ERK. c–d HT29 cells were transfected with RGS2-HA (0.1 μg), RGS4-HA (0.1 μg) or RGS16-HA (0.1 μg) and treated with Fluo-4 dye-loading solution for 1 h. Fluo-4 solution was replaced with Tyrode’s solution containing 60 μM AYPGKF and intracellular calcium levels measured for 2000 s at 10 s intervals (c). Relative intracellular calcium levels measured at 960 s are shown in (d). e Beads charged with bacterially expressed GST-Rhotekin-RBD were incubated with extracts of HT29 cells expressing 10 μg plasmid encoding RGS2-HA, RGS4-HA or RGS-HA16 in the presence and absence of 20 μM AYPGKF. Bound proteins were immunoblotted with anti-RhoA antibodies. HT29 cell extracts (10%) were used as the loading input for the GST pulldown assay and immunoblotted with anti-RhoA and -HA antibodies. Coomassie blue staining was used to estimate the levels of GST-Rhotekin-RBD fusion proteins. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, compared to unstimulated control, and #P < 0.05, ##P < 0.01, ###P < 0.001, ####P < 0.0001, compared to AYPGKF-stimulated control. The results are representative of at least three independent experiments