JAK3 mutants signal through JAK1, STAT5, and ERK in a cytokine-independent manner. (A) Western blot analysis of whole-cell lysates of Ba/F3 cells transformed by JAK3 mutants. Phosphorylation of JAK1 was detected for all JAK3 mutants. JAK3 phosphorylation was clearly detected for some, but not all, JAK3 mutant proteins, most likely because of the specificity of the used antibody. JAK3 protein expression was detected with a human-specific antibody, not recognizing the endogenous JAK3 expression. (B) JAK3 phosphorylation could be detected for all JAK3 mutants after immunoprecipitation and detection with a phosphotyrosine antibody (4G10). (C) All transforming JAK3 mutants were able to phosphorylate downstream signaling components STAT5 and ERK.