Adp hpd

The cell pellets were lysed with Cell Lysis Buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) containing: 1 mM sodium fluoride and 1 mM sodium orthovanadate (phosphatase inhibitors), 250 nM ADP-HPD (Sigma, A0627; a PARG inhibitor to prevent PAR chain cleavage during extraction), 20 μM PJ34 (a PARP inhibitor to prevent PAR synthesis during extraction), 1x phosphatase inhibitor cocktail (Sigma-Aldrich, P0044, P5726) and 1x complete protease inhibitor cocktail (Roche, 11697498001. The lysates were incubated on ice for 30 min with gentle mixing and clarified by centrifugation at 21,000 RCF in a microfuge for 15 min at 4 °C.

Cells were stimulated with H₂O₂ (Sigma Aldrich) for 10 min in PBS at 37 °C, collected by washing with ice-cold PBS and lysed in modified RIPA buffer (50 mM Tris pH 7.5, 400 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 0.1% Na-deoxycholate), protease inhibitor mixture (Roche) supplemented with 2 mM Na-orthovanadate, 5 mM NaF, 5 mM Glycero-2-phosphate, 1 μM ADP-HPD (Millipore) and 40 μM PJ-34 (Enzo Life Sciences) and cleared by high-speed centrifugation. Proteins were precipitated by adding fourfold excess volumes of ice-cold acetone and stored at −20 °C overnight. Subsequently, proteins were solubilized in a urea solution (6 M urea/2 M thiourea/10 mM HEPES pH 8.0). Protein concentrations in lysates were measured using Bradford assay (Bio-Rad). Next, proteins were reduced by adding dithiothreitol to a final concentration of 1 mM, and alkylated with chloroacetamide at 5.5 mM. Proteins were digested using endoproteinase Lys-C (1:100 w/w) and modified sequencing grade trypsin (1:100 w/w) after a fourfold dilution in 50 mM ammonium bicarbonate solution. Protease digestion was terminated by slow addition of trifluoroacetic acid to pH 2. Precipitates were removed by centrifugation for 10 min at 3,000g. Peptides were purified using reversed-phase Sep-Pak C18 cartridges (Waters). Peptides were eluted off the Sep-Pak with 50 and 80% acetonitrile.

Cells expressing the tagged versions of the proteins of interest were collected by washing with PBS and lysed in modified RIPA buffer (50 mM Tris pH 7.5, 400 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 0.1% Na-deoxycholate), protease inhibitor mixture (Roche) supplemented with 2 mM Na-orthovanadate, 5 mM NaF, 5 mM Glycero-2-phosphate, 1 μM ADP-HPD (Millipore) and 40 μM PJ-34 (Enzo Life Sciences). Lysates were diluted in modified RIPA without salt and then cleared by high-speed centrifugation.
GFP-immunoprecipitaion was performed with 20 μl GFP-Trap_A agarose beads (Chromotek). 1 mg of protein mixtures were incubated for 2 h rotating at 4 °C before washing and subsequent elution with 2 × Laemmli sample buffer (Thermo Fisher Scientific) at 90 °C. Pull down of ADP-ribosylated proteins was performed similarly but using 200 μl crosslinked Af1521 macro domain and 2 mg of protein mixtures.