Fig. 2.

Effect of leptin on activation of Akt. Serum-starved OE33 cells were stimulated with leptin 1 nM. Where indicated inhibitors (API-2 1 μm, GSK690693 1 μM) were added 60 min prior to leptin or a dominant-negative Akt adenoviral construct (DN-Akt) added 24 h prior to leptin. (a) Effect on Akt phosphorylation. After 5 min, cells were formalin-fixed and subsequently phosphorylated Akt and total Akt levels quantified in fixed cells using a specific cell-based ELISA. Results for each well were normalised for cell content using subsequent crystal violet staining. Results are expressed as the ratio of phosphorylated/total kinase relative to control-treated cells. (b) Effect on Akt activity. After 5 min, cells were lysed and Akt kinase activity quantified in Akt immunoprecipitates using a specific kinase (K-LISA) kit. Results are expressed as phosphorylating activity compared to untreated controls. Results are expressed as mean ± SEM, N = 5, *P < 0.01 vs control untreated, **P < 0.01 vs leptin-treated