FIG. 6.
The Iκκ complex but not p90rsk mediates the HIV-induced activation of NF-κB. (A) In vitro kinase assay of Iκκ and p90rsk. Immunoprecipitates (IP) from mock-infected (NI), mock-infected and TNF treated (TNF), and HIV-infected (HIV), SFFV-expressing U937 cells were lysed, and the Iκκ complex and p90rsk were immunoprecipitated with anti-Iκκα and anti-p90rsk antibodies, respectively. Immunoprecipitates were analyzed in an in vitro kinase (IVK) assay with recombinant protein IκBα-wt (positions 1 to 54) or IκBα S32/36A (positions 1 to 54) as a substrate (32P-IκBα). The membrane was subsequently stained with Coomassie blue (stained IκBα) or immunoblotted (Immunoblot) with anti-Iκκα, anti-Iκκβ, or anti-p90rsk antibodies. (B) SFFV-expressing, HIV-infected U937 cells were transiently transfected with con-luc (□) or κB-con-luc (■) together with expression vectors for wild-type (WT) or negative dominant (nd) forms of Iκκα or Iκκβ and a TK CAT reporter gene. Luciferase units were normalized to CAT units. The NF-κB luciferase activity of uninfected, SFFV-expressing cells was similar to that of con-luc in HIV-infected cells, and none of the Iκκα or Iκκβ (wt or nd) expression vectors modified the basal level of plasmid κB-luc activity in uninfected cells (data not shown). This experiment is representative of three additional ones. Each transfection point was determined in duplicate, and error bars indicate ± standard deviations.