FIG. 4.

Effect of RNAi knockdown of cellular GEFs on Nef-associated PAK2 activity. (A) Western blot analysis of JTag cells transfected with small interfering RNA oligonucleotides specific for the indicated GEFs or a nonspecific control oligonucleotide (con) for GEF expression levels, as in Fig. 3. Note that endogenous protein levels were analyzed, except in the case of DOCK2, where an expression plasmid for DOCK2.flag was cotransfected with the RNAi oligonucleotides. (B) Nef-associated PAK2 activity following GEF knockdown. JTag T lymphocytes transfected with GEF-specific small interfering RNA or the control oligonucleotide together with the indicated Nef.GFP expression plasmids were subjected to IVKA following anti-GFP immunoprecipitation (IP). (C) Quantification of the Nef-associated PAK2 activity following GEF knockdown. Intensities of autophosphorylated PAK2 signals were quantified relative to the amounts of immunoisolated Nef.GFP. The relative associated PAK2 activity for Nef.GFP was arbitrarily set to 100%. Data are means ± standard errors of the means from at least five independent experiments. Statistical significance is indicated by the P values derived from Student's t test analysis. wt, wild type.