Figure 2. vGPCR induces activation of Rac but not Rho. (A) Activated mutants of all 3 small G proteins (RhoA, Rac1, and Cdc42) can induce NF-κB activation and IL-6 transcription, similar to vGPCR, in COS-7 cells. (B) 293T cells expressing vGPCR or vGPCR R143Q (R143Q) did not demonstrate elevated levels of active Rho in the absence (-) or presence (+) of 50 nM IL-8 (1 minute) with respect to GFP-expressing cells (control). Similar results were obtained using longer exposure to agonist. The DH/PH domain of PDZ-Rho-GEF was used as a positive control. (C) Transcriptional activation through the κB site by vGPCR in COS-7 cells is not inhibited by treatment with C3 toxin. Gα13 was used as a positive control for C3 toxin effects. Data in panels A and C represent the mean ± SEM of triplicate samples from a typical experiment, expressed as fold induction with respect to control. (D) 293T cells expressing vGPCR had elevated levels of active Rac in the absence (-) or presence (+) of 50 nM IL-8 (1 minute). Agonist-dependent vGPCR mutant (R143Q) only induced activation of Rac in presence of IL-8. The constitutively active Rac GEF, truncated TIAM (TIAM1 C1199), was used as a positive control. (E-F) PAE cells transfected with vGPCR and Rac WT had elevated levels of active Rac (E) and demonstrated Rac-like morphology (F). Cells were fixed and stained with phalloidin-specific antibodies to label the actin cytoskeleton. Arrows indicate membrane ruffling (vGPCR and RacQL) or filopodia (Cdc42QL). Pictures are representative of 3 independent experiments. PAE cells transfected with expression vectors for RhoAQL, Rac1QL, or Cdc42QL were used as controls.
Figure 2.

vGPCR induces activation of Rac but not Rho. (A) Activated mutants of all 3 small G proteins (RhoA, Rac1, and Cdc42) can induce NF-κB activation and IL-6 transcription, similar to vGPCR, in COS-7 cells. (B) 293T cells expressing vGPCR or vGPCR R143Q (R143Q) did not demonstrate elevated levels of active Rho in the absence (-) or presence (+) of 50 nM IL-8 (1 minute) with respect to GFP-expressing cells (control). Similar results were obtained using longer exposure to agonist. The DH/PH domain of PDZ-Rho-GEF was used as a positive control. (C) Transcriptional activation through the κB site by vGPCR in COS-7 cells is not inhibited by treatment with C3 toxin. Gα13 was used as a positive control for C3 toxin effects. Data in panels A and C represent the mean ± SEM of triplicate samples from a typical experiment, expressed as fold induction with respect to control. (D) 293T cells expressing vGPCR had elevated levels of active Rac in the absence (-) or presence (+) of 50 nM IL-8 (1 minute). Agonist-dependent vGPCR mutant (R143Q) only induced activation of Rac in presence of IL-8. The constitutively active Rac GEF, truncated TIAM (TIAM1 C1199), was used as a positive control. (E-F) PAE cells transfected with vGPCR and Rac WT had elevated levels of active Rac (E) and demonstrated Rac-like morphology (F). Cells were fixed and stained with phalloidin-specific antibodies to label the actin cytoskeleton. Arrows indicate membrane ruffling (vGPCR and RacQL) or filopodia (Cdc42QL). Pictures are representative of 3 independent experiments. PAE cells transfected with expression vectors for RhoAQL, Rac1QL, or Cdc42QL were used as controls.

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