Ddx17

Example 6

Cells dimerized as described above were collected and spun down prior to crosslinking with 1% formaldehyde at room temperature for 10 minutes followed by glycine quenching. Cells were lysed in 0.1 M Tris pH 7.5, 10 mM potassium acetate, 15 mM magnesium acetate, 1% NP-40 and spun to isolate nuclei, then nuclei were lysed using 0.1 M Tris pH 8, 1% SDS, 10 mM EDTA. Nuclei were sonicated briefly to solubilize material, and SDS was quenched with dilution buffer containing 0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris pH 7.5, and 167 mM NaCl. Protein complexes were immunoprecipitated overnight using the antibodies against HA (Cell Signaling 3724), Flag (Sigma F1804), or DDX5 (Bethyl A300-523A), all at 1:50, and were washed three times with 100 mM Tris pH 9, 100 mM LiCl, 1% NP-40, and 1% sodium deoxycholate. Complexes were eluted by vortexing twice with 1% SDS, 15 mM NaHCO₃ for 15 minutes each time. Elutes were run on SDS-page gels and probed with antibodies against the HA tag (Cell Signaling 3724), Flag tag (Sigma F1804), CTCF (Cell Signaling 2899), SMC1 (Bethyl A300-055A), or DDX17 (Bethyl A300-509A) as indicated, all at 1:1000.