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. 2009 Feb 25;83(9):4652–4669. doi: 10.1128/JVI.02408-08

FIG. 10.

FIG. 10.

EBNA3C forms a ternary complex with p53 and Mdm2 and facilitates p53 ubiquitination by Mdm2. (A and B) Fifteen million MEF cells (p53−/− Mdm2−/−) were cotransfected with Flag-tagged EBNA3C, HA-tagged Mdm2, and Myc-tagged p53 expression constructs. In each case control samples were balanced with empty vector. Cells were harvested at 36 h posttransfection, approximately 5% of the lysed cells were saved as input, and the remainder were IP with 1.5 μg of M2 antibody. Lysates and IP complexes were resolved by 10% SDS-PAGE and subjected to Western blotting (WB) with the indicated antibodies. The same blots were stripped and reprobed with appropriate antibodies. (B) Fifty million BJAB cells and BJAB cells stably expressing EBNA3C (clone 10) were collected and lysed in RIPA buffer, and protein complexes were IP with p53-specific antibody (αp53). Samples were resolved by 8% SDS-PAGE, and Western blotting for the indicated proteins was done by stripping and reprobing the same membrane. (C) Saos-2 cells were transfected with expression plasmids for HA-tagged Mdm2, untagged EBNA3C, and Myc-tagged p53. At 36 h posttransfection, cells were treated with 40 μg/ml CHX for indicated lengths of time. Ten percent of lysates from each sample were resolved by 10% SDS-PAGE. GAPDH blotting was done for the loading control. Western blotting was done by stripping and reprobing the same membrane. Specific p53 bands in each gel were scanned with Odyssey imager software. (D) Saos-2 cells were transfected with expression vectors for HA-tagged Ub, Flag-tagged Mdm2, untagged EBNA3C, and Myc-tagged p53. Myc-tagged p53 was IP with 9E10 antibody from lysates of transfected cells prepared 36 h after transfection and analyzed by immunoblotting with HA-specific antibody. Five percent of the whole-cell extracts of the transfected cells were subjected to SDS-PAGE and immunoblotting with 12CA5 to determine the overall extent of ubiquitination. The same blots were stripped and reprobed with appropriate antibodies as indicated. All panels are representative gels from similar repeated experiments. Numbers at left of panels are molecular masses in kilodaltons.