Effects of CD45 knockdown and overexpression. (A) Knockdown of CD45 increases sensitivity of the human T-ALL cell line KE-37 to IL-7 stimulation. KE-37 cells were electroporated with a CD45 targeting or control siRNA. At 72 hours after electroporation, a fraction of the cells were stimulated with 10 ng/mL IL-7 for 10 minutes. Phosphorylation of STAT5 was assayed by Western blot (left) and quantified (right). (B) Knockdown of CD45 increases JAK/STAT signaling in MOHITO cells transformed by JAK1 or IL-7R gain-of-function mutants. MOHITO cells transformed by activating JAK1 or IL-7R mutants were transduced with a CD45 targeting or control shRNA. Phosphorylation of JAK1 and STAT5 was assayed by Western blot (left) and quantified (right). (C) Knockdown of CD45 increases proliferation of MOHITO cells transformed by a JAK1 gain-of-function mutant. Proliferation of MOHITO cells expressing both the activating JAK1 A634D mutant and either a CD45 targeting or a control shRNA was followed over a period of 72 hours and normalized to the control shRNA. The average ± SEM of 3 repeats is shown. *Statistical significance. (D) Overexpression of CD45 reduces sensitivity of HEK293T cells to IFN-α treatment. HEK293T cells transfected with empty vector or human CD45 (isoform R0) were stimulated with 500 U/mL IFN-α and harvested at different time points. STAT3 phosphorylation was assayed on Western blot and quantified relative to total STAT3.