FIGURE 3.

Leptin elevates phosphorylation of GSK3β and decreases the formation of GSK3β-LKB1-Axin complex. A, MCF7 breast cancer cells were treated with 100 ng/ml leptin for indicated time intervals. Total lysates were immunoblotted using specific antibodies for phospho-GSK3β. The membranes were re-blotted using total GSK3β and actin antibodies as control. The blots are representative of multiple independent experiments. The histogram is the mean of densitometric analysis showing relative density units (RDU) of the Western blot signal for phosphorylated GSK3β normalized to total GSK3β. *, p < 0.005 compared with untreated controls. Leptin treatment increases phosphorylation of GSK3β. B, breast cancer cells were untreated (U) or treated with 100 ng/ml leptin (L) and subjected to immunoprecipitation (IP) analysis using specific antibodies against LKB1 and Axin. The immunoprecipitates were analyzed by immunoblot analysis using anti-GSK3β antibodies. Immunoprecipitation with IgG was used as control. Leptin decreases the formation of the GSK3β-LKB1-Axin complex. C, breast cancer cells were treated with 100 ng/ml leptin alone (L) and in combination with LiCl (L+Li). Untreated cells are denoted with U. Total lysates were immunoblotted using antibodies against phospho-GSK3β and β-catenin. The membranes were re-blotted using total GSK3β and actin antibodies as control. The blots are representative of multiple independent experiments. Combined treatment with leptin and LiCl further increases β-catenin expression. WB, Western blot.