Figure 1

p21/waf1 and cyclin D2 are overexpressed and in a stable kinase complex in HTLV-1 infected cells. (A) One hundred micrograms of total cellular protein from uninfected CEM and infected C81 cells were prepared, separated by reducing SDS-PAGE on a 4–20% gel, and blotted with anti-Tax polyclonal and anti-actin antibodies. The antigen-antibody complex was detected with ¹²⁵I-protein G. The marker is a ¹⁴C-labeled Rainbow (high molecular weight) Marker. Positions are indicated in kiloDaltons. (B) Western blots were performed as described above using anti-cdk4 rabbit polyclonal, anti-cyclin D2 rabbit polyclonal, anti-p21/waf1 rabbit polyclonal and anti-actin goat polyclonal antibodies. (C) C81 and CEM cell extracts (3 mg) were IPed with anti-p21/waf1 monoclonal antibody or no antibody overnight at 4°C. The complexes were precipitated with protein A+G agarose beads and washed with TNE₃₀₀ + 0.1% NP-40. Proteins were then separated by reducing SDS-PAGE on a 4–20 % Tris-glycine gel and transferred onto a PVDF membrane. All lanes in the top panel are western blotted with anti-cyclin D2 antibody. All lanes in the bottom panel are western blotted with anti-cdk4 antibody. NS indicates non-specific bands. (D) C81 and CEM cell extracts (3 mg) were IPed with anti-p21/waf1 mouse monoclonal, anti-cyclin D2 rabbit polyclonal, anti-cdk4 rabbit polyclonal antibodies, or no antibody overnight at 4°C. The complexes were precipitated with protein A+G agarose beads and washed twice with TNE₃₀₀ + 0.1% NP-40, once with TNE₅₀ + 0.1% NP-40, and twice with kinase buffer. Immune complexes were used for in vitro kinase assays using GST-Rb as a substrate. Kinase reactions (shown in the top panels) were separated on a 4–20 % Tris-glycine gel, dried, and exposed to a PhosphorImager cassette. Lanes 1, 5, and 9 are control lanes for C81 IPs and lanes 2, 6 and 10 are control lanes for CEM IPs, (IPs with only protein A+G agarose beads). Lower panels are control WBs for cdk4 and cyclin D2.