Figure 6
Expression of a dominant negative PAK2 mutant inhibits Nef-mediated enhancement of CD25 expression in Jurkat cells stimulated by PHA-P (1 μg/ml). A and B. Jurkat E6.1 cells were transfected with pCDNA3.1 FLAG-PAK2 K278R (PAK2 DN) and passaged in 1 mg/ml G418 for 15 days. 1.5 × 106 parental E6.1 cells and PAK2 DN cells were transduced with 50,000 cpm of RT activity of the indicated pHAGE Nef-IRES-zsGreen vectors. A. At 48 h post-transduction, cells were lysed in 1% NP-40 lysis buffer. 10 μl of anti-FLAG agarose conjugate beads were used to immunoprecipitate FLAG-PAK2 from 1 mg of lysate. Bead bound proteins were eluted in 2X SDS sample buffer, and analyzed by SDS-PAGE/Western blot for PAK2, Nef, and β-tubulin expression. B. 8 h post-transduction, cells were stimulated with 1 μg/ml PHA-P for 18 h and then stained with CD25 APC-Cy7. Cell surface CD25 and zsGreen expression was determined via flow cytometry. Representative plots are labeled with CD25 MFI for zsGreen+ and zsGreen- cells. C. Averages of 3 replicates ± SEM CD25 MFI are shown. * Significantly different from E6.1 transduced with vector control; p < 0.01 (Student's t-test).