Fig. 6

Protein expression of (a) proliferating cell nuclear antigen (PCNA), (b) hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 (HSD3B1), (c) steroidogenic acute regulatory protein (StAR) and (d) cytochrome P450 family 11 subfamily A member 1 (CYP11A1) after 15 h treatment with DHA. Effects of DHA treatment on protein levels were assessed in bovine granulosa cells after 15 h culture in enriched McCoy’s 5A media in presence or absence of DHA 10 or 20 μM. The chemical DMSO alone (1/2000) was used as a negative control due to its solvent activity on DHA. Protein extracts were separated by electrophoresis on 4–12% (w:v) SDS-polyacrylamide gel. After electrotransfer to nitrocellulose membranes, the proteins were probed with anti-PCNA (a), anti-HSD3B1 (b), anti-StAR (c) or anti-CYP11A1 (d) antibodies. The blots were stripped and re-probed with antibodies against Vinculin (VCL). Results of at least five independent experiments are presented. Bands on the blots were quantified and the total protein / VCL protein ratio was calculated. Results are expressed relative to the control as mean ± SEM of five independent experiments for HSD3B1, StAR, CYP11A1 and six independent experiments for PCNA. * indicates significant difference (p < 0.05) and # indicates tendency (p < 0.10)