Phosstop

Cells were lysed with buffer A (100 mM NaCl, 300 mM sucrose, 3 mM MgCl₂, 10 mM PIPES (pH 6.8), 1 mM EGTA, and 0.2% Triton X100, containing PhosSTOP (Merck, Darmstadt, Germany) and Complete Protease Inhibitor Cocktail (Merck, Darmstadt, Germany) for 8 min on ice. Lysates were centrifuged at 5000× g at 4 °C for 5 min to separate the chromatin-containing pellet. Supernatants were obtained as soluble fractions. The pellet was digested with 50 units of Benzonase (Enzynomics, Daejeon, Korea) for 40 min in RIPA buffer (50 mM Tris–HCl (pH 8.0), 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.5% Na deoxycholate, 1 mM PMSF, 5 mM MgCl₂, containing PhosSTOP (Merck, Darmstadt, Germany) and Complete Protease Inhibitor Cocktail (Merck, Darmstadt, Germany)) to extract chromatin-bound proteins. The chromatin-containing fractions were clarified by centrifugation (13,000× g, 4 °C) for 5 min to remove debris. The protein concentration was determined using the Bradford Assay (Bio-Rad, Hercules, CA, USA), and the proteins were analyzed by immunoblotting.

Cell fractionation was conducted as previously described (Vujanovic et al, 2017 (link)), with modifications. Briefly, cells were treated with 200 ng/ml (WI38VA13) or 100 ng/ml (U2OS) illudin S or irradiated with 20 J/m² UV-C following incubation for 3 or 6 h. Collected cells were resuspended in SB1 buffer (50 mM Tris–HCl [pH 8.0], 150 mM NaCl, 1 mM EDTA, 0.1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, Complete Protease Inhibitor Cocktail [Merck], PhosSTOP [Merck], and 2 mM N-ethylmaleimide [NEM; FUJIFILM Wako Pure Chemical]) and centrifuged at 600g for 5 min at 4°C. This extraction was performed twice. The pellet was resuspended into SB2 buffer (50 mM Tris–HCl [pH 8.0], 1 mM MgCl₂, 1 mM phenylmethylsulfonyl fluoride, PhosSTOP, and 2 mM NEM), sonicated, incubated with benzonase (EMD Millipore) at 30°C for 30 min, and centrifuged at 600g for 5 min at 4°C. The pellet was resuspended in SB2 buffer and centrifuged at 4°C. The resulting supernatants were collected as the chromatin fraction and used for immunoblotting analyses.

Cells were lysed in ice-cold 50 mM tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 5 mM EDTA supplemented with PhosSTOP (Merck), cOmplete (Merck), and 1 mM phenylmethylsulfonyl fluoride (PMSF) for 30 min on ice. To determine DPEP1 protein levels, cells were lysed in ice-cold 50 mM tris-HCl (pH 8), 150 mM NaCl, 0.5% sodium desoxychelat, 0.1% SDS supplemented with PhosSTOP (Merck), cOmplete (Merck), 1 mM PMSF, and 100 mM Octyl-β-d-glucopyranosid for 30 min on ice. Insoluble material was removed by centrifugation (14,000g, 30 min, 4°C). Protein concentration was determined using a commercial bicinchoninic acid assay kit according to the manufacturer’s instructions (Thermo Fisher Scientific). Equal amounts of protein (typically 25 μg per lane) were resolved on a 4 to 15% gradient SDS–polyacrylamide gel electrophoresis gel and transferred to a polyvinylidene difluoride membrane (Bio-Rad). After blocking for 1 hour at room temperature, primary antibody incubation was performed at 4°C overnight. Secondary antibodies (anti-mouse, HRP-linked antibody, and anti-rabbit, HRP-linked antibody, Cell Signaling Technology) were applied at concentrations of 1:5000. Proteins were then visualized by enhanced chemiluminescence (Amersham Biosciences).