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. 2003 Jun;23(12):4230–4246. doi: 10.1128/MCB.23.12.4230-4246.2003

FIG. 1.

FIG. 1.

FIG. 1.

FIG. 1.

FIG. 1.

ELL(eCT) is necessary and sufficient to inhibit p53 transcriptional activity. (A) Full-length or truncated mutants of MLL-ELL and ELL. (B) The indicated constructs were transfected into 293T cells (5 μg of each cDNA), and cell lysates were prepared 24 h after transfection. Western blot analysis was carried out with the indicated antibodies. Actin served as loading control. Examples of short and long film exposures are shown. (C) Flag-p53 expression vector (100 ng) was cotransfected into H1299 cells with PG13 luciferase reporter (0.25 μg) in the presence or absence of various MLL-ELL and ELL constructs (1 and 2 μg for both) by using the Lipofectamine method. pRL-TK was used as a transfection efficiency control. Both Firefly (PG13) and Renilla (pRL-TK) luciferase activity was measured 36 h posttransfection by using the Promega dual luciferase system. The relative luciferase activity was calculated as a ratio of the Firefly activity to the Renilla activity and is expressed as the fold induction over the vector control (mean ± the standard deviation [SD] of three independent experiments, each done in duplicate). (D) H1299 cells were Lipofectamine transfected as indicated with Flag-p53 (100 ng), Flag-ELL (1 μg, 100 ng, and 10 ng), and MLL-ELL and MLL-ELLΔCT (2 and 4 μg for both). PG13 and pRL-TK plasmids were also included in the transfection mixture. The cells were lysed 36 h posttransfection, and the luciferase activity was determined as described in panel A (the mean ± the SD of three separate experiments, each done in duplicate, is shown in the upper panel). Proteins in the remainder of cell lysates were resolved by SDS-PAGE, transferred onto nitrocellulose membranes, and analyzed by Western blot with the indicated antibodies (lower panel). Sample numbers on the luciferase activity chart correspond to those on the Western blot. (E) Cell lysate of 293T cells transfected with Flag-ELL (1 μg) or cell lysates from untransfected cell lines as shown were probed with anti-ELL and anti-p53 antibody. Actin levels were examined to ensure equal loading. N, null; WT, wild type; M, mutant. (F) Schematic depiction of ELL functional domains and ELL truncated mutants. (G) cDNA of ELL or its truncated constructs (5 μg) was transfected into 293T cells. pEGFP (0.5 μg) was included as a transfection efficiency control. Cell lysates were prepared 24 h later and analyzed with the indicated antibody. (H) Plasmids (1 μg) as shown in panel D were cotransfected with Flag-p53 (1 μg), along with PG13-Luc and pRL-TK, into H1299 cells by calcium phosphate precipitation. Luciferase activity, which was recorded 36 h posttransfection, is expressed as the fold induction over vector alone and is shown for three independent transfection experiments, each done in duplicate (mean ± the SD).