Figure 2

Alsterpaullone inhibition of cdk2/cyclin A complex in HIV-1 infected cell. A) Equal amount (2 mg) of cytoplasmic proteins from alsterpaullone-treated ACH2 and OM10.1 cells were immunoprecipitated with anti-cyclin A antibody and the cdk2 activity was examined by in vitro kinase assay using histone H1 as a substrate. Alsterpaullone at various concentrations (0.01, 0.1, 0.5, 1, 5, and 25 μM) were used in treatment of cells. The [γ-³²P]-labeled histone H1 was visualized by autoradiography. Alsterpaullone completely inhibits cdk2 kinase activity in infected cells at 0.5 μM (lane 2). B) Similar to panel A, but used extracts from uninfected cells for IP. Alsterpaullone moderately inhibited cdk2 activity in uninfected CEM and Jurkat cells but only at high concentrations (lanes 4-6). C) Effect of low concentration of alsterpaullone in kinase assay. Similar to panel A and B, a low concentration of alsterpaullone (0.5 μM) was used in kinase inhibition studies. Infected (ACH2 and OM10.1) as well as uninfected control (CEM and Jurkat) cell lysates were used for these assays. Lanes 1, 3, 5 and 7 were cells treated with DMSO and lanes 2, 4, 6, and 8 were treated with alsterpaullone. Results are triplicate experiment of using cyclin A IP as the Kinase and histone H1 as the substrate.