Anti ha or anti flag agarose beads

For immunoprecipitation, cells expressing bait proteins were lysed in 1 mL of lysis buffer containing 150 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 1% Triton. The lysates were clarified at 12,000 × g for 15 min to remove cell debris and the supernatants were incubated with anti-HA or anti-FLAG agarose beads (Sigma-Aldrich) overnight at 4 °C. For cross-linking immunoprecipitation, we used anti-HA agarose beads to minimize the adverse impact of cross-linking on the affinity between antibodies and bait proteins. The beads with bound proteins were washed four times with 1 mL of lysis buffer. Finally, the bound proteins were eluted by FLAG or HA peptides and boiled for 5 min in the SDS-PAGE sample buffer containing 60 mM Tris-HCl (pH 6.8), 1.7% (w/v) SDS, 6% (v/v) glycerol, 100 mM dithiothreitol (DTT), and 0.002% (w/v) bromophenol blue. Then the eluted samples were stored at −20 °C prior to further analyses.

Co-IP and western blotting assays were performed as previously described (Pan et al., 2018 (link)). In brief, cells were lysed with the lysis buffer (150 mM NaCl, 50 mM Tris–HCl [pH 7.5], 1 mM EDTA,1% Triton X-100, 0.5% NP-40), plus PMSF and protease inhibitor cocktail for 30 min at 4°C. The cell lysates were clarified by centrifugation at 18,000 g for 30 min at 4°C, then mixed with anti-HA or anti-Flag agarose beads (Sigma) and incubated at 4°C for 4 hr, followed by washing four times with cold lysis buffer and eluting in gel loading buffer. The immunoprecipitated samples were analyzed by SDS-PAGE and detected by western blotting. Quantity One program (Biorad) was used to quantify the western blotting results. The information of antibodies were shown in Key resources table in the supplemental material.

The assay was performed as described previously (18 (link)) with minor modifications. Briefly, 600 μl nuclear extracts (NE) or whole cell extracts (WCE) were pre-cleared by incubation with 50 μl protein A-beads for 1 h at 4°C and the supernatants were incubated with 50 μl anti-HA or anti-Flag agarose beads (Sigma) for 2 h. The beads were washed twice with buffer D (20 mM HEPES–KOH [Ph7.9], 15% glycerol, 0.2 mM EDTA, 0.1% NP-40, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride) containing 0.3 M KCl and then twice with buffer D containing 0.1 M KCl. The precipitates were eluted off the beads by incubation with 0.1 M glycine (pH 2.5) and analyzed by Western blotting with the indicated antibodies.