Figure 3.
Cell cycle regulation of two ISWI complexes in Xenopus egg extracts. (A) XISWI was immunoprecipitated from interphase (lanes 1 and 2) or mitotic HSS (lanes 3 and 4) in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of ISWI peptide and analyzed by immunoblotting with antibodies against XISWI, XACF1, and XWSTF. (B) XISWI was immunoprecipitated from 32P-labeled HSSs as in A, separated by SDS-PAGE and analyzed by autoradiography. (C) Chromatin was assembled in interphase (lane 1) or mitotic HSS (lane 2) and analyzed by immunoblotting with the antibodies indicated. (D) Chromatin was assembled in interphase (lanes 1 and 2) or mitotic HSS (lanes 3 and 4) that had been mock-depleted (lanes 1 and 3) or depleted of XISWI (lanes 2 and 4). Chromatin-associated polypeptides were separated by 7.5% SDS-PAGE and stained with Coomassie blue. The positions of XACF1, XWSTF, and XISWI are indicated by dots. (E) Sperm chromatin was incubated with interphase LSS. After 120 min, recombinant sea urchin cyclin BΔ90 was added to convert the extract into a mitotic state. At the indicated time points, aliquots were taken and chromatin-bound polypeptides were analyzed by immunoblotting using the antibodies indicated.