PMC:6172693 / 50093-51656 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/6172693","sourcedb":"PMC","sourceid":"6172693","source_url":"https://www.ncbi.nlm.nih.gov/pmc/6172693","text":"Western blotting\nHuman U2OS (ATCC HTB-96) and HEK293T (ATCC CRL-3216) cells were plated and grown overnight in DMEM (Sigma-Aldrich) supplemented with 10% FBS (GIBCO) and penicillin-streptomycin (100 U/ml, GIBCO). To induce DNA damage, cells were incubated with 2 mM H2O2 (Sigma-Aldrich) in DPBS with calcium and magnesium (GIBCO) for the indicated times. For PARPi, cells were pretreated with 10 μM Olaparib for 1 hr, and Olaparib was also added to the DPBS solution in case of the induction of DNA damage. Cells were lysed with Triton X-100 lysis buffer (50 mM Tris-HCl pH 8.0, 100 mM NaCl, 1% Triton X-100) at 4°C. Right before use, the buffer was supplemented with 5 mM MgCl2, protease and phosphatase inhibitors (Roche), 1 μM ADP-HPD and 1 μM Olaparib. Benzonase (Sigma) was added to the cell lysates and incubated for 20 min at 4°C. After centrifugation at 14,000 rpm for 15 min, supernatants were collected. Protein concentrations were analyzed by Bradford Protein Assay (Bio-Rad). Proteins were boiled in NuPAGE LDS sample buffer (Invitrogen), resolved on NuPAGE Novex 4%–12% Bis-Tris gels (Invitrogen), and transferred onto nitrocellulose membranes (Bio-Rad) using Trans-Blot Turbo Transfer System (Bio-Rad). Membranes were blocked in PBS buffer with 0.05% Tween 20 and 5% non-fat dried milk for 1 hr at room temperature, and incubated overnight with primary antibodies at 4°C, followed by 1-hour incubation with peroxidase-conjugated secondary antibodies at room temperature. Blots were developed using ECL (Invitrogen) and analyzed by exposing to films.","divisions":[{"label":"Title","span":{"begin":0,"end":16}}],"tracks":[]}