FIG. 2.

Detection of PR/GR and SRC proteins on the MMTV promoter by ChIP. (A) Ligand- and receptor-mediated recruitment of SRC-1, SRC-2, and SRC-3 was determined by ChIP assays using the same batch of T47D/CAT0 cells treated with progesterone (lanes P) or dexamethasone (lanes D) or untreated cells (lanes −). The lower bands were amplified by using primers for the MMTV promoter, and the slower-migrating bands represent GAPDH control. (B) SRC antibody specificities were tested by using in vitro-translated SRC-1, -2, or -3 or T47D whole-cell lysate by immunoprecipitation-Western blotting with the indicated antibodies. About 20% of each sample was used for inputs. (C) Xenopus oocytes lysate expressed Flag-tagged SRC proteins were prepared in serial twofold dilutions for Western blot analysis using anti-Flag antibody (left panel). Equivalent amounts of each Xenopus lysate dilution were used in Western blotting with anti-SRC-1, -2, or -3. Similar serial dilutions of T47D cell lysate were prepared for Western blotting together with the blots containing Flag-tagged SRC proteins. The bands labeled with asterisks represent nonspecific reactions. Results of Western blotting were quantitated with NIH Image version 1.62. The relative levels of SRC proteins were calculated by normalization with quantitated Flag-SRC references. The relative level for SRC-3 was arbitrarily set as 100%.