JAK1 and JAK3 mutants cooperatively activate the JAK-STAT pathway. (A) A total of 2 × 106 BW5147 cells nontransduced (NT) or stably transduced with JAK1 (WT or Y652H mutant) together with JAK3 (WT, V674A, or R657H mutants) were lysed and subjected to western blot analysis. The basal phosphorylation level of STAT and JAK proteins was detected using anti-pY694 STAT5, anti-pY705 STAT3, anti-pY1034/1035 JAK1, and anti-pY980/981 JAK3 antibodies. Cells expressed equivalent levels of JAK1 and JAK3, as shown with anti-JAK1 and anti-JAK3 antibodies. Membranes were reprobed with anti-STAT3, anti-STAT5, and anti–β-actin antibodies as loading control. (B) A total of 2 × 106 CMK cells NT or stably transduced with JAK1 (WT or A634D mutant) were lysed and subjected to western blot analysis. Basal phosphorylation level of STAT and JAK proteins was detected using anti-pY694 STAT5, anti-pY705 STAT3, anti-pY1034/1035 JAK1, and anti-pY980/981 JAK3 antibodies. Membranes were reprobed with anti-STAT3, anti-STAT5, anti-JAK1, anti-JAK3, and anti–β-actin antibodies as loading control. (C) HEK293 cells were transiently cotransfected with JAK1 (WT or Y652H mutant), JAK3 (WT, V674A, or R657H mutants), the IL-9Ra chain, and common γ-chain, in addition to the STAT5-responsive luciferase reporter construct pLHRE and the pRLTK plasmid as transfection control. Twenty-four hours posttranfection, cells were lysed and subjected to dual luciferase activity assay. Histograms are means ± standard error of the mean of 5 independent experiments. A 1-way analysis of variance test was performed to compare the condition with the 2 mutants vs each condition with 1 mutant only (**P < .01).
