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

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Copyright © 2003, American Society for Microbiology

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FIG. 5. — MLL-ELL and ELL disrupt p53 interactions with p300 transcriptional coactivator. (A) H1299 cells were transfected with the following constructs as indicated: Flag-p53 (0.3 μg), MLL-ELL (2, 5, and 10 μg), MLL-ELLΔCT (2 and 5 μg), and HA-p300 (1 μg). pEGFP (0.5 μg) served as a transfection efficiency control. Empty vector was used to correct for total DNA amount. Cell lysates were prepared 24 h posttransfection and Western blot analysis was carried out with the indicated antibody. (B) A similar experiment was carried out with 1, 2.5, and 5 μg of ELL and with 1 and 2.5 μg of ELLΔeCT. (C) Flag-p53 plasmid (25 ng) was cotransfected into H1299 cells with PG13 luciferase reporter and with or without Flag-ELL (250 ng) and HA-p300 (2 and 4 μg) as indicated. pRL-TK was included to control for transfection efficiency. Values shown are the fold induction in luciferase expression over the vector control (mean ± the SD of three separate experiments). (D) Gal4 and VP16 fusions of p300 and p53. (E) Protein expression of constructs depicted in panel D was analyzed by transfection of 293T cells, followed by Western blot with anti-Gal4 as described. (F) The indicated plasmids, as well as pGL3-G5SV and pRL-TK plasmids, were cotransfected into H1299 cells in the presence or absence of 2 μg of Flag-ELL or Flag-ELLΔeCT. The luciferase activity measured 36 h posttransfection is expressed as the fold induction over the vector control (mean ± the SD of three independent experiments). (G and H) The indicated Gal4 fusions (G), the protein levels of which were examined by Western blot (H), were transfected into H1299 cells with or without Flag-ELL or Flag-ELLΔeCT and luciferase reporters (pGL3-G5SV and pRL-TK). (I) The luciferase expression was measured as described 36 h after transfection and is shown as the fold induction compared with vector control in three separate transfection experiments (mean ± the SD). (J) The indicated p300 Gal4 and ELL VP16 constructs were cotransfected with pGL3-G5SV and pRL-TK into H1299 cells. (K) The luciferase activity was assessed 36 h posttransfection (the fold induction over the vector control is shown as the mean ± the SD of three separate experiments, each done in duplicate).

FIG. 5. — MLL-ELL and ELL disrupt p53 interactions with p300 transcriptional coactivator. (A) H1299 cells were transfected with the following constructs as indicated: Flag-p53 (0.3 μg), MLL-ELL (2, 5, and 10 μg), MLL-ELLΔCT (2 and 5 μg), and HA-p300 (1 μg). pEGFP (0.5 μg) served as a transfection efficiency control. Empty vector was used to correct for total DNA amount. Cell lysates were prepared 24 h posttransfection and Western blot analysis was carried out with the indicated antibody. (B) A similar experiment was carried out with 1, 2.5, and 5 μg of ELL and with 1 and 2.5 μg of ELLΔeCT. (C) Flag-p53 plasmid (25 ng) was cotransfected into H1299 cells with PG13 luciferase reporter and with or without Flag-ELL (250 ng) and HA-p300 (2 and 4 μg) as indicated. pRL-TK was included to control for transfection efficiency. Values shown are the fold induction in luciferase expression over the vector control (mean ± the SD of three separate experiments). (D) Gal4 and VP16 fusions of p300 and p53. (E) Protein expression of constructs depicted in panel D was analyzed by transfection of 293T cells, followed by Western blot with anti-Gal4 as described. (F) The indicated plasmids, as well as pGL3-G5SV and pRL-TK plasmids, were cotransfected into H1299 cells in the presence or absence of 2 μg of Flag-ELL or Flag-ELLΔeCT. The luciferase activity measured 36 h posttransfection is expressed as the fold induction over the vector control (mean ± the SD of three independent experiments). (G and H) The indicated Gal4 fusions (G), the protein levels of which were examined by Western blot (H), were transfected into H1299 cells with or without Flag-ELL or Flag-ELLΔeCT and luciferase reporters (pGL3-G5SV and pRL-TK). (I) The luciferase expression was measured as described 36 h after transfection and is shown as the fold induction compared with vector control in three separate transfection experiments (mean ± the SD). (J) The indicated p300 Gal4 and ELL VP16 constructs were cotransfected with pGL3-G5SV and pRL-TK into H1299 cells. (K) The luciferase activity was assessed 36 h posttransfection (the fold induction over the vector control is shown as the mean ± the SD of three separate experiments, each done in duplicate).