FIG. 5.

F30 can be reconstituted in a heterologous cell system. (A) 293T cells were individually transfected with expression vectors for Flag-Maml, N^ic-Myc, or HA-CSL, and nuclear lysate was fractionated on a Superose 6 size exclusion column. Transfected proteins are indicated on the left, and the antibodies used for immunodetection are listed on the right. (B) N^ic and Maml form F30 with endogenous CSL in 293T cells. Cells were cotransfected with expression vectors encoding Flag-Maml and N^ic-Myc, and nuclear lysate was processed as before. Each transfected protein was detected by Western blotting as previously described. Endogenous CSL was detected with anti-CSL polyclonal antisera. (C) Formation of F30 by cotransfection of N^ic, Maml, and CSL in 293T cells. Cells were cotransfected with expression vectors encoding Flag-Maml, N^ic-Myc, and HA-CSL, and nuclear lysate was fractioned by size exclusion chromatography. Each transfected protein was detected by Western blotting as previously described. (D) N^ic, Maml, and CSL are physically associated in F30 when transfected into 293T cells. Ten percent of the column load (Input) and three fractions covering the indicated complex peaks (F30 and F37) were individually pooled and immunoprecipitated with αN^ic-927 (lanes IP) or IgG control (lanes C). Proteins were detected by Western blotting with the indicated antibody. (E) ΔRAM incorporates into F30 in 293T cells when coexpressed with Maml and CSL. 293T cells were cotransfected with expression vectors encoding Flag-Maml, ΔRAM-Myc, and HA-CSL, and proteins were processed and detected as previously described.