Fig. 5. TIS7 can associate with the SIN3 complex. (A) mSin3B, HDAC1, TIS7 and SAP30 partially co-fractionate. Nuclear extracts prepared from control (–E2) or 4 day E2-treated c-JunER cells (+E2) were fractionated on a 10–35% continuous sucrose gradient. Aliquots from 0.6 ml fractions were resolved on a 10% SDS–PAGE gel and immunoblotted with the indicated antibodies. In mSin3B western blot, the arrowhead indicates the full-length mSin3B LF band; other bands detected by this antibody, mainly in lighter fractions, represent degradation products. Sedimentation coefficients of 14 and 20S fractions were marked according to the peaks of proteasome and cohesin proteins used as markers. (B) c-JunER cells were either left untreated (–E2) or cultured in the presence of 10^–6 M E2 for 4 days. Nuclear extracts were prepared as described in Dignam et al. (1983), and immunoprecipitated with the affinity-purified α-TIS7 antibody. Co-immunoprecipitated endogenous proteins were detected by western blotting with the indicated antibodies. The additional band in the TIS7 western blot is an as yet unidentified post-translational modification present in E2-treated cells.