JAK3 FERM domain mutations show increased kinase activity in vitro. (A) Direct determination of the kinase activity for FERM domain mutations using the Lance Ultra TR-FRET–based assay. Background counts were seen in assays without ATP addition, for BaF3 cells, and with immunoprecipitations using isotype control IgG. Recombinant human JAK3 (50 pg) was used as a positive control. Specific immune complexes of WT and mutant JAK3s showed kinase activity with ATP. The counts are shown as the average of 9 independent kinase assays (mean ± SEM). Statistical comparison between mutant JAK3s and WT JAK3 showed significantly higher kinase activity in the mutants (*P < .001). WT JAK3 showed statistically significant increased activity compared with IgG control (P = .008). (B) Shows Western blots of JAK3 from a representative immunoprecipitation and the detection of autophosphorylated JAK3 after in vitro assay. 4G10 is specific for phosphotyrosine and the third blot shows a phospho-Y980 of JAK3. Notably, a kinase-dead compound mutant E183G/ K855A showed no autophosphorylation.