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. 2004 Dec;24(23):10366–10380. doi: 10.1128/MCB.24.23.10366-10380.2004

FIG. 6.

FIG. 6.

(A) H1299 cells were transfected with pCB6+p53, pCDNA-VRK1-myc, pCMV-p300-HA, or pCMV-Chk2 as indicated. The transfected p53 was immunoprecipitated using monoclonal antibodies, and the blot was analyzed for the acetylation of p53 on Lys 373 and Lys382 with a specific antibody. IP, immunoprecipitation; IB, immunoblotting. (B) VRK1 promotes the formation of a p53-p300 complex. H1299 cells were transfected with different combinations of the indicated plasmids (pCMV-p300-HA, pCB6+p53, and pCDNA-VRK1-myc). Extracts from transfected cells were immunoprecipitated with an anti-p300 polyclonal antibody, and the blot was developed with either anti-HA to detect immunoprecipitated transfected p300 or anti-p53 to detect the coimmunoprecipitated p53. (C) H1299 cells were transfected with pCB6+p53, pVRK1-HA, and p3.1MdmX-myc as indicated, and the levels of p53 were determined with a mix of anti-p53 antibodies. Quantification of the amount of p53 is shown below. (D) The H1299 cell line was transfected with plasmid encoding p53 with a substitution of Thr18 for alanine (1 μg) with or without pCDNA-VRK1-myc (8 μg), and 36 h postransfection, whole-cell extract was subjected to immunoblotting to detect p53 total protein. Six hours prior to extract collection, MG132 was added to the cells where indicated.