Figure 6
Effect of insulin after 24-h stimulation with rh adiponectin on phosphorylation of MAPK ERK 1/2 in bovine granulosa cells. Granulosa cells from small follicles were cultured for 24 h in enriched McCoy's 5A medium supplemented with 10% FBS, starved of serum for 24 h and then incubated in serum-free medium with or without rh adiponectin (10 μg/mL) for 24 h. Then, granulosa cells were cultured in fresh serum-free medium with or without insulin (10-8 M) for 5 min. Protein extracts were separated by electrophoresis on 12% (w:v) SDS-polyacrylamide gel. After transfer to nitrocellulose membranes, the proteins were probed with anti-phospho-ERK1/2. The blots were stripped and reprobed with antibodies against vinculin. Bands on the blots were quantified and the phospho-ERK1/2/vinculin ratio was calculated. Values represent means ± SE relative to the basal state from three independent experiments. Bars with different superscripts are significantly different (P < 0.05).