Figure 1.

Tyrosyl phosphorylation of wild-type and Tyr→Phe Ob-Rb constructs in 293T cells. (A) Tyrosyl phosphorylation of wild-type Ob-Rb after leptin binding in transfected 293T cells. Cells were treated with leptin and were lysed, and Ob-Rb was immunoprecipitated. The immunoprecipitate was blotted with an antiphosphotyrosine monoclonal antibody (pY20). Total receptor expression was scored by immunoblotting of the same filter with an anti c-myc monoclonal antibody that recognizes the c-myc tag fused in frame to Ob-Rb. (B) Tyrosyl phosphorylation of Tyr→Phe Ob-Rb mutants. 293T cells transfected with JAK2 and different Ob-Rb mutants were treated with leptin (as above). Receptor phosphorylation and expression was confirmed with pY20 and the anti c-myc monoclonal antibody. A schematic diagram of wild-type and Tyr→Phe Ob-Rb constructs is shown below.