Fig. 5.
Treatment of PC12 cells with exogenous Tat and HIV-1 infected culture media inhibits p35/Cdk5 kinase activity. Approximately 2 × 106 U-937 cells grown in RPMI with 2% fetal bovine serum were infected with HIV-1 JF-RL and after 5 days post-infection, supernatants were collected and used for incubation with PC12 cells. Approximately 2 × 106 PC12 cells maintained in media containing NGF were treated with exogenous Tat protein or conditioned media obtained from infected U-937 cells. Protein extracts were harvested, immunoprecipitated with p35 antibody, and analyzed by kinase assay using purified histone H1 as a substrate. A decrease in the intensity of the band corresponding to the phosphorylated H1 was observed in Tat-treated PC12 cells (lane 2) and after treatment of cells with conditioned media from HIV-1 infected macrophages (lane 3). Lane 4 represents PC12 cells treated with conditioned media from uninfected monocytes. Lane 5 represents the negative control, non-immune mouse serum (NS) used in the precipitation step. Densitometric analysis data for the p35 kinase assay are presented. The kinase assay was performed twice and the results were normalized to total H1 input and presented in arbitrary units.