Figure 5

Increased levels of cyclin E/cdk2 kinase activity in HTLV-1 infected cells. (A) Seventy-five micrograms of total cellular protein from CEM and C81 cells were prepared, separated by SDS-PAGE on a 4–20% Tris-glycine polyacrylamide gel, and blotted with anti-cyclin E rabbit polyclonal antibody or anti-cdk2 rabbit polyclonal antibody. (B) C81 and CEM cells extracts (3 mg) were IPed with anti-cyclin E polyclonal antibody overnight at 4°C. The complexes were precipitated with protein A+G agarose beads, washed with TNE₆₀₀ + 0.1% NP-40 twice, and then with kinase buffer twice. The IP's were then used for in vitro kinase assays using histone H1 as a substrate and varying incubation times of 5, 30 and 45 minutes at 37°C. Kinase reactions were processed as described in the methods section. The bottom panel shows a coomassie blue staining of the gel. (C) Relative amounts of kinase activity as determined using the ImageQuant software. (D) Kinase assays were performed as described above using histone H1 as the substrate. H9 are uninfected T-cells and Hut 102 are HTLV-1 infected cells.