Functional characterization of residues to the N-terminal side of the Nef polyproline helix: V
, and F
. (A), In vitro PAK2 autophosphorylation assays were performed on anti-Nef immunoprecipitates from transfected 293T cells. Upper Panel, Autoradiography shows the autophosphorylated PAK2 band, indicated by the arrow. Lower Panel, Nef expression was determined by anti-Nef Western blot of whole cell lysates. One of two experiments is presented. (B), MHCI downregulation and CD4 downregulation by Nefs from (A). Upper Panel, Flow cytometric analysis of Nef-transduced human CEM T cells was performed to assay cell surface expression of CD4 (x-axis) and MHCI (y-axis). Lower Panel, Quantitation of MHCI downregulation relative to SF2Nef was set at 100%. Error bars calculated as the mean ± S.E.M. of three independent experiments. An asterisk indicates a significant reduction in activity relative to SF2Nef.