Ne per nuclear extraction kit

Cell fractionation was performed using the NE-PER nuclear extraction kit (Thermo Scientific) according to manufacturer’s instructions. RNA was extracted using the previously mentioned protocol.

For endogenous immunoprecipitation experiments, nuclear extracts from the prostate cancer cell lines LNCaP and VCaP were obtained using NE-PER nuclear extraction kit (Thermo Scientific). Nuclei obtained were lysed in the IP buffer (20 mM Tris-HCl, pH 7.5, 1% Triton-X, 150 mM NaCl and complete protease inhibitor cocktail (Roche)). Nuclear lysates (0.5-1.0mg) were then pre-cleaned by incubation with protein G Dynabeads (Life Technologies) for 1 hour at 4°C. 5μg antibody was added to the pre-cleared lysates and incubated on a rocker at 4°C overnight prior to the addition of protein G Dynabeads for 1hr. Beads were washed thrice in IP buffer, resuspended in 40 μL of 2× loading buffer and boiled at 90°C for 10 minutes for separation of the protein and beads. Samples were then analyzed by immunoblotting as described above.
To assess the effect of MI-136 on interaction between MLL complex and AR, VCaP cells were treated with varying concentrations of MI-136 for 24 hrs and nuclear extracts were prepared as described above. Immunoprecipitation was performed using anti-menin antibody exactly as described above followed by immunoblotting using anti-AR and anti-MLL antibody.