Figure 1
From: Myc interacts with Max and Miz1 to repress C/EBPδ promoter activity and gene expression
Myc represses C/EBPδ promoter activation. A. c-Myc (Myc), C/EBPδ, Cyclin D1, Miz1, Max and Sp1 protein levels in HC11 nontransformed mouse mammary epithelial cells under exponentially growing (GR) and growth arrest (GA) conditions (Western blot). B. Western blots from "A" were scanned to assess relative Myc, C/EBPδ, Cyclin D1, Miz1, Max and Sp1 protein levels in HC11 cells under Growing (Grow) vs Growth Arrest (GA) conditions. Due to differences in antibody affinity, quantitative comparisons are only valid for individual proteins under Grow vs GA conditions. C. HC11 cells were co-transfected with increasing amounts of a V5 tagged Myc expression construct (5-50 ng) plus a C/EBPδ promoter luciferase reporter construct. C/EBPδ promoter driven luciferase results were normalized to co-transfected Renilla luciferase control activity. Luciferase results from Myc treated cells are expressed relative to the vector control results, which were set as "1". D. Whole cell lysates (20 ul) from (C.) were immunoblotted and probed with an anti-V5 antibody to assess V5-tagged Myc protein levels in vector control (grey bar) and Myc transfected (black bar) HC11 cells. E. Schematic representation of Myc full length and Myc deletion mutants. Full length c-Myc contains: Myc box 1 (MB1, 45-63aa), MB2 (129-143aa), nuclear localization signal (NLS, 320-328aa), basic region (BR, 355-367aa), helix-loop-helix (HLH, 368-410aa) and leucine zipper (LZ, 411-439aa). F. HC11 cells were co-transfected with a C/EBPδ promoter luciferase reporter construct plus full length Myc or Myc deletion mutant expression constructs (V5 tagged). C/EBPδ promoter driven luciferase activities were normalized to co-transfected renilla luciferase control activity. C/EBPδ promoter driven luciferase results from Myc constructs are expressed relative to the vector control results, which were set as "1". G. Whole cell lysates from luciferase assays in (D.) were immunoblotted and probed with an anti-V5 antibody to assess Myc and Myc deletion mutant protein levels. All luciferase results shown are the average-fold changes relative to the vector control values from 2-3 independent experiments with duplicates performed in each experiment.