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FIG. 7.

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Acetylation of ATDC K116 is critical for p53-ATDC interaction. (A) For the top panel, 293T cells were cotransfected with equal amounts (4 μg) of HA-ATDC with or without expression plasmids for Flag-PCAF or HA-p300. Cell lysates were immunoprecipitated (IP) under high-stringency conditions using anti-HA antibodies. Immunoprecipitates were subjected to Western blot (IB) analysis using anti-acetyl-lysine (AcK) antibodies. The blot was stripped and reprobed with the indicated antibodies to confirm equal immunoprecipitation efficiency and loading. For the lower panel, HA-ATDC, which was expressed and purified from 293T cells, was digested with trypsin and subjected to ion trap mass spectrometry (ITMS). The positions of the unambiguously identified acetylated lysine residues are shown. (B) The top panel shows an alignment of amino acids 102 to 116 of ATDC from different species. Residue 116 is highlighted in red. For the bottom panel, lysates prepared from U2OS cells expressing wild-type or mutated HA-ATDC were immunoprecipitated with anti-HA antibodies and Western blotted with anti-p53 or anti-HA antibodies. (C) Representative images of U2OS cells transfected with plasmids expressing either wild-type or mutated ATDC, fixed, stained with antibodies, and analyzed by confocal microscopy. (D) For the top left panel, cell survival assays were performed with AT5BIVA cells transfected with plasmids that express wild-type or mutant HA-ATDC. In the top right and bottom left panels, pBP100-GL2 or p21Luc reporter plasmids were transfected into U2OS cells, with or without plasmids expressing wild-type or mutant HA-ATDC. The luciferase activity was determined 24 h after transfection. The results from the averages of three independent experiments ± the SD are shown. For the bottom right panel, Western blots were performed to assess comparable expression of wild-type versus mutant ATDC. (E) Extracts prepared from U2OS cells transfected with the pcDNA3.1HA vector or stably expressing HA-ATDC or the HA-ATDC K116R mutant, treated or untreated by IR, were subjected to Western blot analysis to examine the effect of ATDC acetylation/deacetylation mutation on p53-mediated p21 expression.

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