Nef-Pak2 complex proteins are expressed at comparable levels in pHAGE-Nef transduced Jurkat cells utilized for mass spectrometry. Proteins immunoblotted in 1% of the input lysates utilized for LC-MS/MS analysis are shown. Jurkat E6-1 cell lysates were harvested 48 h after transduction with VSV-G pseudotyped pHAGE-Nef-HA-IRES-ZsGreen vector virions encoding wild-type wild-type (Lane 2 and 6) or mutant Nefs (Lanes 3, 4, and 7), or empty vector (Lanes 1 and 5). Lysates analyzed in Lanes 5–7 were derived from unsorted cells, while lysates in Lanes 1–4 were from cells sorted to obtain populations with matched ZsGreen fluorescence. β-tubulin is included as a loading control.