Mouse igg beads

Cells grown in E2-deprived medium were treated with E2 (10⁻¹¹ M), 4-OHT (10⁻⁷ M), or the vehicle (ethanol) for 4 hrs before being isolated for whole cell or nuclear extracts. To conduct IP using whole cell extracts, cells were lysed in RIPA buffer supplemented with 1 mM PMSF and protease and phosphatase inhibitors (Roche Applied Science). The cell lysates were precleared with mouse IgG beads (Sigma, 3 hours at 4°C) before NF1 (mAb-376) or ER (F-10 monoclonal, Santa Cruz) antibody or the mouse IgG control (Santa Cruz) was added. Samples were incubated at 4°C overnight, followed by incubation with protein A/G agarose beads (Santa Cruz) for another 4 hours. All beads were finally washed three times with TBS (20 mM Tris-HCl (pH 7.5) and 150 mM NaCl). The bound proteins were eluted with 2× SDS sample buffer by boiling for 5 minutes and then examined by immunoblotting. To conduct IP using nuclear extracts, the same antibodies were pre-incubated with the protein-G Dynabeads™ (Thermo Fisher Scientific) overnight at 4°C before mixing with nuclear extracts which were prepared as described previously (Lanz et al., 2010 (link)). All beads were washed twice with NETN buffer (20 mM Tris-HCl (pH 7.5), 1 mM EDTA, 150 mM NaCl, and 0.5% NP-40), and once with PBS before elution and immunoblotting.