Figure 2

Attenuation of LEPRb signaling is not due to receptor internalization and depends on active gene expression. A, Binding of leptin-SEAP to RINm5F cells stably expressing LEPRb was determined after cells were treated with leptin (20 ng/nl) for 2 h or were not treated (Ctrl). Nonspecific binding was determined by incubating parallel cultures with leptin-SEAP in the presence of excess unlabeled leptin (2 μg/ml) and was subtracted. Bound phosphatase activity was determined using a chemoluminescence-based assay. Bars represent means and S.D. values of six independent experiments. Reduction of LEPRb surface expression after treatment with leptin was not significant (paired t-test). B, RINm5F cells stably expressing wild type LEPRb were treated with actinomycin D (5 μg/ml) for 30 min before stimulation with leptin (20 ng/ml). The time course of leptin-induced STAT3 phosphorylation was analyzed by Western blots of total cellular lysates with the indicated antibodies. This results are representative of two independent experiments.