p53 And p65 interact in HTLV-I–transformed cells. (A) Nuclear extracts were prepared from a number of lymphocytic cell lines as indicated and p53 immunocomplexes were isolated. Western blot analysis was then performed to assess the level of associated p65 protein (top panel). IgG antibody was used as a control for specificity. The middle panel indicates the amount of p53 immunoprecipitated. The lower panel indicates the amount of p65 in each extract. (B) Nuclear extracts from HTLV-I–infected cells C81, Hut102, and MT2 as well as the control cell line Molt4 were immunopreciptiated with anti-p53 sera and washed extensively, and proteins were separated on 10% Tris-glycine gels. Western blot analyses for p65, p50, c-Rel, and p53 were performed. (C) Western blot analysis of nuclear extracts (25 μg) from cells used in panel B indicates the level of expression of p65, p50, c-Rel, p53, and Tax. (D) Nuclear extracts from Molt4 cells 4 hours after treatment with 8 Gy γ-irradiation, MG132 (50 μM), or untreated or from MT2 cells were prepared, and immunocomplexes were isolated with control IgG or p53 (Ab6) antibodies and separated on 10% Tris-glycine gels (Invitrogen). Proteins were transferred to PVDF membranes, and p65 levels were visualized (top panel) with anti-p65(A) (Santa Cruz Biotechnology). The lower panel indicates the amount of p53 immunoprecipitated (Ab7; Oncogene Research Product).