Figure S5. Signaling pathways in bovine granulosa cells after treatment with other concentrations of DHA (50 μM) or TUG-891 (10 and 50 μM). Effects of DHA or TUG-891 on phosphorylation of (A) mitogen-activated protein kinase 14 (MAPK14), (B) AMP-activated protein kinaseα (AMPKα) and (C) mitogen-activated protein kinase 1/3 (MAPK1/3) signaling pathways were assessed in bovine granulosa cells cultured for 15 h in enriched McCoy’s 5A media with 50 μM DHA or with 10 or 50 μM TUG-891, as described in Material and Method section for 5, 10, 30 and 60 min. 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-phosphorylated (p-)MAPK14 (A), anti-p-AMPKα (B) or anti-p-MAPK1/3 (C) antibodies. The blots were stripped and re-probed with antibodies against MAPK14, AMPKα, or MAPK1/3, respectively. Bands on the blots were quantified. Results of four independent experiments are presented as the ratio of p-protein to total protein, normalized by the ratio observed in control at each time and expressed as mean ± SEM, with time 0 min being equal to 1 (for reference). Bars with different superscripts are significantly different (p < 0.05). (TIF 99 kb
