FIGURE 5.

Effect of SecinH3, Wortmannin and Gallein on ARF6 activation in HCG-stimulated HEK-HLHCGR cells. HCG-stimulated (10 IU/ml HCG, 30 min) ARF6 activation was assessed by a GST-GGA3 PBD pulldown assay in HEK-HLHCGR cells preincubated with the indicated concentrations of (A) SecinH3, (B) Wortmannin, or (C) Gallein. Total ARF6 levels in the cell lysate and the resin bound ARF6-GTP were visualized by immunoblot analysis with an anti-ARF6 mouse mAb. Densitometric analysis of ARF6-GTP is shown as a histogram after normalizing to the expression of total ARF6 present in the sample. For Wortmannin and Gallein, the effect of the drug vehicle (0.5% DMSO) on ARF6-GTP levels, was also determined. Solid bars indicate measurements made in the presence of HCG.