FIG. 6.

ACTR interacts directly with E2F1 in vivo and in vitro through the N-terminal regions of ACTR and E2F1. (A) ACTR-E2F1 complex in T-47D cells was analyzed by coimmunoprecipitation assay with cells treated with ICI and infected with adeno-ACTR (labeled with an A) or adeno-GFP (labeled with a G). Two days after infection, cell lysates were prepared and immunoprecipitated with α-E2F1 or α-mouse IgG. The presence of ACTR and E2F1 in the immunoprecipitates was detected by Western blotting using α-ACTR and α-E2F1 antibodies. Input was equivalent to 20% of the cell lysate used in the IP. (B) 293T cells were cotransfected with expression plasmids for ACTR and E2F1 or Flag-E2F4 as indicated. Whole-cell lysates were immunoprecipitated with α-ACTR or α-E2F4, followed by Western analysis with α-ACTR, α-E2F1, or α-Flag antibodies. The input represents 20% of the cell lysates. (C) In vitro direct interaction between ACTR and E2F1 was analyzed by GST pull-down assay using purified proteins. Purified Flag-ACTR proteins were incubated with either GST, GST-E2F1, or GST-E2F4 immobilized on glutathione beads. After extensive washing, proteins retained on beads were separated by SDS-PAGE and detected by Western analysis using α-Flag antibody. Input represents 30% of Flag-ACTR used in the assay. (D and E) A GST pull-down assay was also performed with in vitro-translated and ³⁵S-labeled full-length or fragments of ACTR proteins and GST or GST-E2F1. Labeled proteins retained on the beads after washing were analyzed by SDS-PAGE and autoradiography, together with 10% of the translated products used in the incubation. Note that the Gal4 DNA-binding domain was fused to small ACTR fragments to enhance their in vitro translation signals. (F) GST fusions of full-length and fragments of E2F1 were incubated with in vitro-translated N-terminal fragments of ACTR. The left panel shows Coomassie stain of GST and the GST-E2F proteins used.