Turnover of ectopically expressed Myc proteins in Burkitt's and non-Burkitt's cells.
(A), Stability of FLAG-epitope tagged wild type (wt) c-Myc in Raji Burkitt's lymphoma cells. 5 μg of pcDNA3-FLAG-Myc-wt (FLAG-wt c-Myc) or pcDNA3 (Mock) were electroporated into 2 × 107Raji cells together with 15 μg of carrier DNA. 16 hours after electroporation aliquots of cells were treated with CHX for the indicated times or left untreated. FLAG-tagged wt c-Myc was immunoprecipitated from the cell extracts with M2 FLAG antibodies, followed by immunoprecipitation of the endogenous mutated c-Myc protein from the same extracts with IG-13 α-Myc serum. The immunoprecipitated material was then subjected to immunoblot analysis using C33 Myc antibodies. Note that the exposure time of the film presented at the left α-FLAG panel is longer for than for the right α-Myc panel. (B, C), pulse chase analysis of v- and c-Myc turnover in U-937 monoblasts stably expressing the OK10 v-Myc protein (B) and of wt c-Myc and the T58A mutant after transient transfections of U2OS osteosarcoma cells (C). The pulse chase was performed at the indicated time points as described in the legend to Figure 3. In (C), semiconfluent U2OS osteosarcoma cells were transfected with 2 μg of CMV-driven c-myc per 10-cm dish. The pulse chase was performed 16 hours after transfection.
