Parp 1 antibody f 2

The total protein of the extracted cells was lysed in ice-cold cell lysis buffer (NEB) containing 1 mM PMSF and 1:100 protease inhibitor cocktail (Sigma). The lysate was pre-cleared with 15 × l protein A Sepharose 4B beads (Sigma) at 4°C for 30 min. NE-PER^TM Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, 78833) were used to extract nucleoprotein from cells. The BCA protein assy (Pierce, Rockford, IL, United States) and Western Blot procedures were performed as described previously (Ruiz et al., 2019 (link)). Antibodies used included Anti-beta Actin antibody (1:50000, Abcam, ab49900), Anti-gamma H2A.X (phospho S139) antibody (1:1000, Abcam, ab2893), PARP-1 antibody (F-2) (1: 500, Santa Cruz, sc-8007), Cdc2 p34 antibody (17) (1:500, Santa Cruz, sc-54), BRCA1 antibody (D-9) (1:500, Santa Cruz, sc-6954), Phospho-cdc2 (Tyr15) Antibody (1:1000, Cellsignal, #9111), Phospho-BRCA1 (Ser1497) Polyclonal Antibody (1:1000, Thermo Fisher Scientific, # PA5-64621), Rad51 Antibody (G-5) (1:500, Santa Cruz, sc-133089), XRCC1 (1:1000, Abcam, ab44830), and Lamin B1 (1: 20000, Proteintech, 66095-1-Ig).

Total proteins isolated with the AllPrep DNA/RNA/Protein Mini Kit were quantitated using the BCA Protein Assay Kit (Thermo Scientific Pierce, Grand Island, NY, USA). The Human PARP1 DuoSet ELISA kit and the DuoSet Ancillary Reagent kit from R&D Systems (Minneapolis, MN) were used to perform the ELISA assays as per manufacturer’s instructions [27 (link)]. Extracted protein samples were prepared at a 1:20 or 1:50 dilution for the assay. We used PARP-1 antibody (F-2), catalog number sc-8007 from Santa Cruz Biotechnology. The epitope is for amino acids position 764–1014 at the C-terminus of PARP1 human origin. Recombinant human PARP1 standard provided with the ELISA kit was used to create a seven-point standard curve. A reagent blank was included in the ELISA assays, consisting of the reagent diluent provided in the ancillary reagent kit with no protein sample added. Each standard, sample and reagent blank were run in duplicate when performing the ELISA. Results of the ELISA assays were read using a SpectraMax M5 microplate reader (Molecular Devices, LLC, San Jose, California) at a wavelength of 450 nm, and analyzed using SofMaxPro software v. 6.5.1 (Molecular Devices, LLC, San Jose, California) provided with the microplate reader.

Western blotting was performed as described previously 16 (link) using nuclear extracts or whole cell lysates. The following primary and secondary antibodies were used. The primary antibodies were mouse monoclonal c-MYC antibody (9E10; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), goat polyclonal ABCB5 antibody (46-620; ProSci Inc., Poway, CA, USA), goat polyclonal MRP5 antibody (Abcam, Cambridge, MA, USA), rabbit monoclonal BCRP/ABCG2 antibody (Abcam), rabbit monoclonal PCNA antibody (D3H8P XP; Cell Signaling, Danvers, MA, USA), mouse monoclonal PARP1 antibody (F-2; Santa Cruz Biotechnology, Inc.) and mouse monoclonal α-Tubulin antibody (B-7; Santa Cruz Biotechnology, Inc.). The secondary antibodies were antimouse immunoglobulins conjugated to horseradish peroxidase (Igs-HRP), anti-rabbit Igs-HRP and anti-goat Igs-HRP (Dako, Carpinteria, CA, USA).