Figure 3. Activation of Gαi by GIV terminates Arf1 signaling.

(A) Depletion of GIV promotes accumulation of COPI vesicles in the ER-Golgi intermediate compartment (ERGIC). Left: Control (Ctrl siRNA; upper panel) or GIV-depleted (GIV siRNA; lower panel) HeLa cells were fixed and stained for β-COP (red) and ERGIC-53 (green). Merged panels display the overlay of β-COP (red) and ERGIC-53 (green) panels. Insets on merge panels viewed at higher magnification shows limited colocalization of ERGIC-53 with β-COP in the cis-Golgi in control cells and that ERGIC-53 is also found in vesicles scattered throughout the cytoplasm which do not stain for β-COP. In GIV-depleted cells there is increased colocalization between β-COP and ERGIC-53 and that these vesicular structures that co-stain for both β-COP and ERGIC-53 are more dispersed. The white line in the insets indicates the pixels used for the RGB profile plots shown (see Experimental Procedures). Bar = 10 μm. Right: RGB profiles of ERGIC-53 (green) and β-COP (red) corresponding to lines in merge of (A) as shown in insets. (B) COS7 cells were treated with control (−) or GIV siRNA (+), and adenoviral vectors expressing siRNA-resistant GIV-WT or GIV-FA as indicated. Equal aliquots of lysates (bottom panels, 5% input) were analylzed for Arf1•GTP by carrying out GST pulldown assays with GST-GGA3. Bound proteins (top panel) were analyzed for active Arf1 by immunoblotting. (C) Bar graphs display the band densitometry quantification of Arf1•GTP (bound)/total Arf1 (inputs) in B. Data was normalized to control and expressed as % changes. Results are expressed as mean ± S.E.M. (D) COS7 cells expressing Gαi3-WT, Gαi3-WF mutant, or control vector were analyzed for levels of Arf1•GTP as in B. (E) Bar graphs display the band densitometry quantification of Arf1•GTP (bound)/total Arf1 (inputs) in D. Results are expressed as mean ± S.E.M.