Fig. 4.

Effect of inhibition of small GTP-binding proteins on leptin-stimulated Akt activity. OE33 cells were transfected with expression plasmids containing DN-Ras, DN-RhoA, DN-Rac1 or DN-cdc42. After 24 h serum-starvation, cells were stimulated with 1 nM 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 < 0.05 vs leptin-treated, ***P < 0.01 vs leptin-treated