JAK1 kinase activity is essential for the transforming properties of JAK3 mutants. (A) Western blot detection of JAK3 mutants expressed in 293T cells. (B-C) Western blot analysis of whole-cell lysates after reconstitution of the IL7-receptor signaling complex in 293T cells. The 293T cells were transiently transfected with the constructs as indicated. (D) The graph shows relative proliferation of Ba/F3 cells expressing JAK3 M511I, R657Q, or L857Q 48 hours after knockdown of endogenous Jak1 or Jak2 compared with scrambled siRNA. Rescue of the proliferation of cells with Jak1 knockdown was achieved by expression of human JAK1, but not by expression of kinase dead JAK1. Relative proliferation is shown 48 hours after knockdown of endogenous Jak1. Knockdown efficiency was determined by quantitative polymerase chain reaction for all cell lines; results are shown for one cell line but are representative of all cell lines.