FIG. 5.

Regulation of Myc turnover by p300/CBP in HEK293 cells. (A) Cells were transfected with Flag-Myc wt and, as indicated, with expression vectors for p300, CBP, CBPΔHAT, or the empty vector (−). Cycloheximide (CHX) was added at time zero and whole-cell extracts were prepared at the indicated times (in minutes), and Flag-Myc (arrowhead) was detected by Western blotting with the Flag antibody. An asterisk indicates vinculin detected on the same blot with a specific antibody, as a loading reference. (B and C) Same as in panel A, but cells were incubated with HDAC inhibitors for 8 h prior to (and during) treatment with cycloheximide. In panel C, Flag-Myc wt and mutant R6 were analyzed with or without cotransfected p300. (D) As above, but Flag-Myc wt was immunoprecipitated with the Flag antibody, and approximately equal amounts of immunoprecipitated Flag-Myc were loaded on the gel (equal. load) and analyzed by Western blotting by successively probing with the Acetyl-K antibody (Ac-Myc) and then with the Flag antibody (Total Myc). (E) Same as in panel D except that MG-132 was present during cycloheximide treatment. (F) The half-lives (T1/2) of Flag-Myc wt and R6 mutant without and with cotransfected p300, CBP, and CBPΔHAT were determined in the absence (−) and presence of HDAC inhibitors and normalized to the half-life of Flag Myc wt without cotransfected HAT which was set to 1* (average Myc wt T1/2 ± the SD indicated in minutes). The relative T1/2 is the average (±the SD) from at least three independent experiments. The value in parentheses indicated by a superscript “a” is from one experiment. n.a., not analyzed.