FIG. 4.

CARM1 and Tax directly interact in vitro and in vivo. (A and B) GST or GST-Tax wild type (A, lanes 1 and 2) and GST-Tax deletion mutants (B) were incubated with purified CARM1 protein. Shown is Western blot analysis of CARM1 levels from GST-Tax pull-down after incubation of HeLa cell nuclear extract with GST or GST-Tax (A, top panel, lanes 3 and 4). Samples were separated by electrophoresis in 4 to 20% Tris-glycine gels, and Western blot analysis for CARM1 or GST was performed. (C) Nuclear extracts from C81 cells were immunoprecipitated (IP) with anti-CARM1 antibody (Ab) and washed extensively, and proteins were separated by electrophoresis in 4 to 20% Tris-glycine gels. Western blot analyses for Tax, CARM1, or p300 were performed. (D) Colocalization of Tax and CARM1. C81 cells were fixed and then immunostained with anti-Tax (α-Tax) and/or anti-CARM1 (α-CARM1) antibodies. (E) 293T cells were transfected with Tax WT or mutant del 151-204, M29, M30, or M32, using Effectene transfection reagent (QIAGEN). At 48 h posttransfection, cell lysates were prepared and immunoprecipated with anti-CARM1 antibody. Immunoprecipitates were separated by electrophoresis in 4 to 20% Tris-glycine gels. Western blot analyses for Tax or CARM1 were performed. The lower panel indicates the levels of expression of Tax and CARM1. (F) Luciferase activities of HTLV-1 LTR were measured in 293T cells transfected with Tax WT or mutants.