FIG. 5.
Assay for the chromatin binding of transcription factors. (A) Quiescent C10 cells were stimulated with medium containing 10% FBS for 1 h, and the nuclei were purified as described in Materials and Methods. After cell lysis, soluble cytoplasmic and unbound nuclear proteins (fraction S1) were removed and pelleted nuclei were washed. The washed nuclei (P1) were then split into two aliquots, with one incubated in the presence of DNase I and the other incubated under the same conditions without the addition of the nuclease. Material not solubilized by DNase I was then recovered by centrifugation (P2 fraction), and supernatants (S2 fraction) were removed. The distribution of c-Fos and Akt was determined by Western blotting of the protein from the equivalent of 5 × 105 cells or nuclei from each fraction. (B) Nuclei were prepared from either serum-starved C10 cells (lanes 1 to 3) or S-phase cells 20 h after serum stimulation (lanes 4 to 6), and the amount of chromatin-bound (S2 fraction) versus bound Rb (P2 fraction) was analyzed by immunoblotting. (C) Quiescent cells were stimulated with medium containing 10% FBS with or without the addition of SIN-1 (1 mM). Chromatin fractionation was performed with the equivalent of 5 × 105 cells, and the recovery of c-Fos in cytoplasmic (or free) fractions (S1) and nuclease-soluble chromatin fractions (S2) was analyzed by immunoblotting.