Figure 7

Nef residues important for enhancement of viral replication and cell activation are also important for association of Nef with PAK2 in co-precipitation assays. A. 293T cells were transfected with 0.5 μg pCR3.1 Nef-HA or empty vector, 0.5 μg pCDNA.31 FLAG-PAK2 K278R, and 0.5 μg CDC42 V12 in 6-well plates. 48 h post-transfection, cells were lysed in 1ml 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, separated on a 12% polyacrylamide gel, and transferred to PVDF. Expression was detected by Western blot.