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. 2004 Jul 19;101(30):10919–10924. doi: 10.1073/pnas.0404278101

Fig. 1.

Fig. 1.

Testosterone induces phosphorylation of CREB and ERK in primary rat Sertoli cells. (A) Sertoli cells were stimulated with ethanol (vehicle), estradiol (E2), and testosterone (T) at the concentrations shown or ovine FSH (100 ng/ml) for 15 min. Western immunoblotting of whole cell extracts was performed sequentially with antisera specifically recognizing CREB phosphorylated on serine 133 (P-CREB), P-ERK1/2, and all forms of ERK. Fractionation and probing of the samples was repeated by using an antiserum recognizing all forms of CREB. P-CREB and P-ERK1/2 levels were normalized to overall CREB and ERK1/2 expression, and the normalized P-CREB and P-ERK1/2 levels for vehicle-treated cells were arbitrarily set equal to 1. The mean fold induction (±SE) of hormone-stimulated P-CREB (▪) and P-ERK1/2 (□) over vehicle-treated cells for five (P-CREB) and four (P-ERK1/2) independent experiments is reported in the graph to the right. Values that are significantly different from vehicle-treated controls (P < 0.05) are indicted with an asterisk (*). (B) Sertoli cells were stimulated with ethanol for 15 min (0) or 100 nM R1881 for the times shown. Western analyses were performed as in A. The mean (±SE) relative levels of P-CREB (▪) and P-ERK1/2 (□) normalized for CREB and ERK1/2 expression are provided for four (P-CREB) and six (P-ERK1/2) independent experiments with statistical analysis as described in A. No changes in protein expression were observed over 12 h with vehicle (EtOH) controls (data not shown). (C) Sertoli cells were treated for 60 min with EtOH (vehicle), estradiol (E2), RU 5020, or testosterone (T). Western analyses were performed for P-CREB and CREB. The data shown are representative of three independent experiments. (D) Sertoli cells were pretreated with DMSO, actinomycin D (Actino D, 50 μg/ml), or puromycin (10 μg/ml) for 2 h before stimulation with EtOH vehicle (–) or 100 nM testosterone (+). Western blot analyses of P-CREB and CREB levels are shown. The blot shown is representative of at least three experiments.