Figure 2
From: Overlapping effector interfaces define the multiple functions of the HIV-1 Nef polyproline helix
Mutational analysis of the HIV-1 Nef-SH3 interaction domain: PQVPLR, P 78 , and F 90 . (A) Upper Panel in vitro PAK2 autophosphorylation assay of anti-Nef immunoprecipitates from transfected 293T cells. Autoradiography shows the autophosphorylated PAK2 band, indicated by the arrow. Higher molecular weight bands that are weakly phosphorylated are proteins associated with the Nef/activated PAK2 complex [43]. Control represents transfected 293T cells without Nef expression. Middle Panel, Anti-Nef Western blot of whole cell lysates demonstrates equal Nef expression in transfected cells. Lower Panel, Phosphoimager quantitation of activated PAK2 relative to SF2Nef set at 100%. Error bars were calculated as the mean ± S.E.M. of three independent experiments. An asterisk indicates a significantly lower value compared to SF2Nef by t test (p < 0.05). SF2NefV74I and SF2NefP78G are not significantly different from SF2Nef by t test (p = 0.065 and p = 0.065, respectively). (B) Upper Panel, flow cytometric analysis of SF2Nef transduced human CEM T cells. The cell surface expression of CD4 (x-axis) and MHCI (y-axis) was determined as described in Methods. Control represents CEM cells transduced without Nef expression. Lower Panel, Quantitation of MHCI downregulation relative to SF2Nef is presented. SF2Nef, n = 6, is set at 100%. Error bars were calculated as the mean ± S.E.M. P72G, n = 6; Q73R, n = 4; V74I, n = 4; P75G, n = 6; L76V, n = 4, R77K, n = 4; P78G, n = 3; F90A, n = 3. An asterisk indicates a significantly lower value from SF2Nef by t test (p < 0.05). Only P78G is not significantly higher than control by t test.