Figure 5.

Effect of hCG on the localization of Arf6 in granulosa cells. Preovulatory rat granulosa cells were treated with vehicle or 1 IU hCG for 40 min and then subjected to IF using anti-Arf6 antibody or to subcellular fractionation. (A) Distribution of Arf6 in vehicle and hCG-treated cells (a and b) was evaluated by IF using confocal microscopy. Results are representative of five separate experiments for each treatment. Edge staining was greatly reduced by incubating antibody with 500 μg of synthetic peptide overnight at 4°C before addition to fixed cells (c). (B) Western blots were performed on supernatant (S) and pellet (P) fractions using anti-Arf6 antibody. Protein load of supernatant fraction was ≈90 μg; pellets were resuspended in the same volume as supernatants and load volume was that of the corresponding supernatant. Results are representative of two separate experiments. Rat follicular membranes, like porcine follicular membranes, exhibit GTP- and β-arrestin-dependent LH/CGR desensitization (data not shown).