Figure 5

Effect of SOCS3 knockdown on reporter gene induction by LEPRb point mutants. A, SOCS3 expression levels. - HEPG2 cells were transfected with the expression plasmids for the indicated LEPRb constructs, pSUPER-SOCS3 (300 ng/well) or pEF-SOCS3 (10 ng/well). Levels of endogenous SOCS3 in leptin-treated cells (16 h, 100 ng/ml) or overexpressed FLAG-SOCS3 were determined by immunoprecipitation and Western blot analysis with SOCS3-specific antibody. B and C, Knockdown of SOCS1 or SOCS3. The reporter gene assays were performed as in Figure 3 except that shRNA vectors specific for SOCS1 or SOCS3 were co-transfected (300 ng/well). Columns reflect luciferase activities relative to the activity in cells transfected with empty knockdown vector (means ± S.D., n = 3 independent experiments in B and n = 5 in C). The difference of the responses of FYY and FFY to the knockdown of SOCS3 was significant (Student's t-test, P < 0.05).