Fig. 2. Cell cycle-dependent recruitment of HDAC-1 and histone H4 deacetylation on DHFR promoter: compilation of results from Figure 1. Chromatin from synchronized NIH 3T3 cells, either in G₁ phase (gray bars) or at the G₁–S transition (black bars), was analyzed by the XChIP procedure, followed by quantitative PCR of eluted DNA. Equal amounts of chromatin (A) were subjected to immunoprecipitation using anti-HDAC-1 (B) or anti-AcH4 (C) antibodies. DHFR or GAPDH sequences (as indicated) were detected by quantitative PCR. Copy numbers were estimated by reference to a plasmid containing the promoter sequence and used as a standard; (A) number of copies in the inputs; (B) and (C) fraction of the total number of copies detected as antibody-bound material; a control sample run in parallel in the absence of antibodies revealed little association of DHFR (0.01% of the input) or GAPDH (0.002% of the input). Shown are the results of a typical experiment, with range bars indicating standard deviations of triplicates. This experiment has been reproduced four times with similar results. (D) Chromatin, immunoprecipitated by anti-HDAC-1 (+) or control (–) antibodies or before immunoprecipitation (input), was incubated for 15 min at 95°C and analyzed by western blotting, using an antibody that recognizes HDAC-1, HDAC-2 and HDAC-3 (H80920; Transduction Laboratories).