Mitochondrial fusion could allow access of ERK into mitochondria. A.

<p>MA-10 cells were treated with or without CCCP (5 µM) and 8Br-cAMP (1 mM) for 1 h, or incubation with CCCP and 8Br-cAMP for 1 h followed by washout and 3 h recovery. Mitochondria were isolated and western blotting was performed. An image of a representative western blot is shown (Top panel). Membranes were sequentially blotted with anti-ERKt and anti-III Complex antibodies. For each band, the OD of the expression levels of ERKt protein was quantified and normalized to the corresponding III Complex protein. The relative levels of ERKt protein are shown. Data are presented as an average SEM of three independent experiments. a <i>P</i><0.001 vs. cAMP and b <i>P</i><0.01 vs. control. <b>B.</b> Left panel: MA-10 cells were transfected with the shRNA1 SHP2 plasmid. After 48 h, mitochondria were isolated and western blotting was performed. An image of a representative western blot is shown. Membranes were sequentially blotted with anti-ERKt (total ERK) and anti-III Complex antibodies. For each band, the OD of the expression levels of ERKt protein was quantified and normalized to the corresponding III Complex protein. The relative levels of ERKt protein are shown. Right panel: an image of a representative western blot is shown. Membranes were incubated with a stripping buffer and blotted with anti-pERK (phosphorylated ERK). For each band, the OD of the expression levels of pERK protein was quantified and normalized to the corresponding III Complex protein. The relative levels of pERK protein are shown. The results are expressed as the means ± SEM of three independent experiments. a <i>P</i><0.01 vs. Control and b <i>P</i><0.01 vs. 8Br-cAMP mock. <b>C.</b> After 24 h transfection with mt-YFP, MA-10 cells were treated with or without PD98059 (50 µM) and stimulated with hCG (20 ng/ml) or 8Br-cAMP (1 mM) for 1 h. Cells were scored and mitochondrial shapes classified. The results are expressed as the means ± SEM of three independent experiments. ns <i>P</i>>0.05 vs. cAMP and hCG without inhibitor. Cellular medium was used to determine P4 production by RIA. Results of P4 measurement are indicated as the means ± SEM of three independent experiments. ***<i>P</i><0.001 vs. cAMP without PD. *<i>P</i><0.05 vs. hCG without PD.