Journal: Endocrinology

Article Title: Rapid nongenomic E2 effects on p42/p44 MAPK, activator protein-1, and cAMP response element binding protein in rat white adipocytes.

doi: 10.1210/endo.143.3.8678

Figure Lengend Snippet: FIG. 5. Effect of various upstream component inhibitors of MAPK signaling pathway on E2 activation of MAPK in rat adipocytes. A, In vitro-differentiated parametrial adipocytes were pretreated with or without PTX (100 ng/ml) for 16 h and then incubated for 5 min with E2 (10 nM) or LPA (100 M). B, In vitro-differentiated parametrial adipocytes were pretreated with the Src family kinase inhibitor PP2 (10 M) or the vehicle (DMSO 0.1%) for 1 h and then incubated for 5 min with E2 (10 nM) or LPA (100 M). B (inset), In vitro-differentiated parametrial adipocytes were incubated for 5 min with E2 (10 nM) or LPA (100 M). Membrane fractions were prepared as described in Materials and Methods and were analyzed by Western blots using a specific antibody recognizing phospho-Src (Tyr 416). Representative Western blot analysis from one experiment repeated three times are presented. C, In vitro-differentiated parametrial adipocytes were pretreated with wortmannin (100 nM) or the vehicle (DMSO 0.1%) and then incubated with E2 (10 nM) or insulin (100 nM). D, In vitro-differentiated parametrial adipocytes were pretreated for 30 min with the tyrosine kinase inhibitor Genistein (50 M), MEK inhibitor UO 186 (10 M), PKC inhibitor GFX 109203X (0.5 M), or the vehicle (DMSO 0.1%) and then incubated with E2 (10 nM) for 5 min. Cell lysates were analyzed as described in Materials and Methods. Results are expressed as arbitrary units. Each bar represents the mean SEM of five separate experiments. *, P 0.05 by t test, prevention by inhibitor of MAPK activation.

Article Snippet: The phospho-CREB (Ser 133) and phospho-Src (Tyr 416) antibodies were obtained from New England Biolabs, Inc. (Beverly, MA).

Techniques: Activation Assay, In Vitro, Incubation, Membrane, Western Blot