Anti ha antibody conjugated beads

HeLa cells at 70–80% confluency, transfected with the indicated plasmids or treated with H₂O₂, were washed with 1 × phosphate-buffered saline (PBS) and resuspended in RIPA buffer (1 × PBS, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS) along with 1 × protease inhibitor cocktail and phosphatases inhibitor cocktail (Roche Applied Science). To detect PML SUMOylation in HeLa cells, whole-cell extracts in the presence of NEM were prepared. Lysed cells were centrifuged at 4°C at 12 000 r.p.m. for 10 min, and the supernatant was incubated with protein A-conjugated beads for preclearing. To detect protein–protein interactions or acetylation and SUMO1 modification of PML, whole-cell extracts were incubated with anti-HA antibody-conjugated beads (Sigma F2426) or anti-FLAG antibody-conjugated beads (Sigma E6779) for 2 h. The beads were washed with NETN buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 10% glycerol, 1 mM dithiothreitol, and 0.1% NP-40) five times, and supernatants were discarded. Then, 2 × sample buffer was added to the beads, followed by SDS-PAGE and western blotting as previously described.^{9 (link), 10 (link)}

Cell lyses for protein extraction and co-immunoprecipitation were performed as follows. Equivalent number of cells to OD₆₀₀ = 5 was harvested by centrifugation at 3200 × g at 4 °C for 5 min. Pellets were washed with 4 ml TM buffer (10 mM Tris-HCl pH 7, 10 mM MgCl₂) and subsequently frozen at -20 °C to facilitate cell lysis. Cell pellets were then resuspended in 1 ml ice-cold lysis buffer (50 mM Tris-HCl pH 7.5, 5 mM MgCl₂, 250 mM NaCl) supplemented with cOmplete mini protease inhibitor (Roche) and 10 U/ml RNasin^® (Promega). Cells were lysed by sonication and lysates cleared by centrifugation at 16 100 × g for 30 min at 4 °C. Cell lysates were treated with 10 U Turbo™ DNase I (Roche) at 37 °C for 15 min prior to immunoprecipitation. Fifteen μl of anti-Flag^® or anti-HA antibody-conjugated beads (both mouse monoclonal, Sigma) were washed 3 times with TBS (50 mM Tris-HCl pH 7.4, 150 mM NaCl) and added to cell lysates and incubated at 4 °C with rotation overnight. Beads with precipitated RNP-complexes were washed 3 times with 0.5 ml TBS and finally resuspended in 400 μl TBS. RNA was extracted from RNP-complexes by two subsequent phenol/chloroform/isoamyl alcohol (25:24:1) extractions and ethanol-precipitated with NaOAc and glycogen as a carrier. RNA integrity was assessed by analyses of 2 ng of co-immunoprecipitated RNA by a Agilent RNA 6000 Pico Kit.