HR′CMV p12^Itransduction in primary PBMCs increases STAT5 activation.

(A) The HR′CMV p12^I and HR′CMV-Luc constructs were transfected in 293T cells and protein extracts subjected to immunoprecipitation and Western blot analysis using αHA1 Ab. (B) Confocal immunofluorescence of PBMCs after infection with pseudotype viruses expressing either Luc or p12^I. (C) Increased STAT5 DNA binding was indicated by EMSA. STAT5 DNA binding activity was measured by using a ³²P-labeled β-casein GAS element. B indicates basal STAT5 activity; S, starved for serum and IL-2 overnight; minus sign, no Ab to STAT5 after pulse with 1000 U IL-2; plus sign, Ab to STAT5 was added; and 1000, pulse with 1000 IU IL-2. Cells were pulsed with various amounts of IL-2 (0, 0.1, 1, 10, and 100 IU/mL) as indicated. (D) Western blot analysis using a phospho-STAT5–specific Ab was done on the same cellular extracts used in the gel-shift assay.