(A) STAT5 depletion decreases BCL10 mRNA expression in a dose dependent manner. Kit225 cells were treated with antisense STAT5 (AS STAT5) or control (CTRL) ODN as indicated and cultured for 24 h. Cells were harvested in duplicates. One pellet was used for total RNA isolation and cDNA preparation as described in the Methods. QRT-PCR was performed with primers specific to human BCL10. (B) STAT5 depletion decreases BCL10 protein expression. Parallel cell pellets were lysed and equal amounts (10 μg) of lysates resolved on 10% SDS-PAGE, then Western blotted with antibodies to BCL10, STAT5, followed by re-probing with antibodies to GAPDH (as a loading control). Representative data from two independent experiments are shown. (C) STAT5 depletion decreases Kit225 cell viability. Cell viability following electroporation (24 h) was assessed using MTS assay as described in the Methods. Cells were cultured either in the absence (white bars) or presence (black bars) of IL-2. Representative data from three independent experiments are shown. (* p < 0.001) (D) Antisense STAT5 treatment decreases NFκB DNA binding. Kit225 cells were electroporated with either AS STAT5 (AS STAT5) or control ODN (CTRL) and cultured in medium without (-) or with (+) IL-2 for 24 h. Nuclear proteins were isolated and incubated with [32P]-labeled NFκB probe (indicated to the right). Corresponding un-labeled NFκB probe in 100-fold molar excess (lane e) or free probe (lane f) was used to confirm the binding specificity. % DNA-binding was calculated by normalizing band intensities of STAT5 ODN treated samples to the corresponding CTRL ODN treated samples (100%). Representative data from two independent experiments are shown.