Figure 3. Cooperation between Myc Boxes (MBs) is required for amplifier activity.

(A) Summary of mutations introduced into the MBs of transfected MYC. B-H. Effect of various mutations in MBs, individually or in combination, on MYC amplifier activity. Total Renilla or Luciferase activity with EtOH or high Dex concentrations (50 or 100 nM) in U2OS cells, transiently transfected with 2 ng of GR plasmid, 100 ng GREtkLUC reporter, and the indicated amounts of MYC plasmid, were determined as described in Materials and methods, normalized to that for 10 ng plasmid, averaged, and plotted ± SEM (n = 2, 3, or 4) for B) wild-type, (C) MBI mutation, (D) MBII mutation, (E) MBIII mutation, (F) MBIV mutation, (G) MBI+III mutation, and H) MBI+IV mutation. R² values for polynomial fits of Dex/Luc curves were all ≥0.96 except for E and F, which were fit to smooth curves. (I). MYC protein levels (immunoblot) of transfected wild-type (WT) and mutant plasmids. (J) Inverse relationship between MYC protein level and amplifier activity for wild-type and mutant proteins. Data are averages ± S.E.M. from 2 to 5 experiments (n = 9 for wt MYC).