Mutants derived from patients 2 and 3 lack JAK3 catalytic activity. COS-7 cells transfected with the indicated cDNAs were lysed and immunoprecipitated with anti-JAK3 antibody. In vitro kinase assays of JAK3 autophosphorylation were performed at room temperature on the immunoprecipitates for 1 or 5 minutes as indicated. Samples were then resolved by SDS-PAGE, transferred to nitrocellulose, and subjected to autoradiography to visualize³²P incorporation (top blot). The membrane was probed with anti-JAK3 Ab to assess expression level and gel loading of the various JAK3 proteins (bottom blot).