F2426

Cells were lysed with lysis buffer containing 50 mM Tris-HCl (pH 7.8), 150 mM NaCl, 1% NP-40, 0.1% deoxycholate and protease inhibitor cocktails (Roche). For DNase and RNase treatment, cell lysates were treated with 40 U ml⁻¹ DNase and 10 μg ml⁻¹ RNase at 37 °C for 30 min. Lysates were immunoprecipitated using anti-Flag (Sigma-Aldrich, F2426) or anti-HA (Santa Cruz, sc-805 AC) antibodies. For co-immunoprecipitation of endogenous proteins, E13.5 mouse brains were homogenized in the above lysis buffer. Lysates were immunoprecipitated using TLX antibody, followed by immunoblotting using indicated antibodies. To determine whether Dpi disrupts the TLX-Drosha interaction, constructs expressing Dpi (TLX residues 341–359) or a control peptide (TLX residues 201–223), together with HA-TLX and Flag-Drosha were transfected into HEK293T cells. Cell lysates were immunoprecipitated with Flag antibody (Sigma-Aldrich, F2426), followed by immunoblotting with anti-HA (1:500, Santa Cruz, sc-805) or anti-Flag antibody (1:500, Sigma-Aldrich, F1804). Images in Figs 1a,b, 2c,d,f and 5b–e have been cropped for presentation. Full size images are presented in Supplementary Figs 8–10.