Figure 6

Assay of JAK2 activity in LEPRb complexes. A, Outline of the method. - RINm5F cells stably expressing WT or mutant (FFY) LEPRb were treated with leptin (20 ng/ml) for the 15 min or 120 min as indicated. LEPRb receptor complexes were immunoprecipitated (IP) with an antibody directed against the C-terminal myc tag of LEPRb. Immunoprecipitates were incubated with recombinant STAT1 as substrate in the presence of ATP, and phosphorylation of STAT1 was determined by Western blot analysis of the supernatant (kinase activity). B, Western blot analysis. - Activation loop phosphorylation of JAK2 was assessed in the immunoprecipitates with a phosphospecific antibody (anti Tyr1007/1008, PY-JAK2). Catalytic activity of receptor-bound JAK2 was determined as the phosphorylation of STAT1 on Tyr701 (PY-STAT1). LEPRb and total STAT were detected to control for comparable levels of expression or immunoprecipitation, respectively. A control reaction that was performed in the absence of ATP is marked by an asterisk (*). In two independent experiments, STAT1 phosphorylation by LEPRb-WT complexes was reduced by 57.3% and 42.6% after 2 h of leptin treatment, whereas the STAT1 phosphorylating activity of LEPRb-FFY complexes remained constant (103.1% and 111.3%).