Protein Extraction and Analysis from Cells

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).

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Li H., Wang C., Lan L., Wu W., Evans I., Ruiz E.J., Yan L., Zhou Z., Oliveira J.M., Reis R.L., Hu Z., Chen W., Behrens A., He Y, & Zhang C. (2021). PARP1 Inhibitor Combined With Oxaliplatin Efficiently Suppresses Oxaliplatin Resistance in Gastric Cancer-Derived Organoids via Homologous Recombination and the Base Excision Repair Pathway. Frontiers in Cell and Developmental Biology, 9, 719192.

Publication 2021

protease inhibitor cocktail protein A Sepharose 4B beads NE-PERTM Nuclear and Cytoplasmic Extraction Reagents Anti-beta Actin antibody Anti-gamma H2A.X (phospho S139) antibody PARP-1 antibody (F-2)

Anti antibody Antibodies Beta actin Brca1 Buffer Cdc2 Cells Cold Cytoplasmic Gamma H2a x Lamin b1 Nucleoprotein Parp 1 Protease inhibitor Protein Protein a sepharose Western blot Xrcc1

Corresponding Organization : The First Affiliated Hospital, Sun Yat-sen University

Other organizations : University of Oklahoma Health Sciences Center, University of Minho

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Variable analysis

independent variables

Cell lysis buffer (NEB) containing 1 mM PMSF and 1:100 protease inhibitor cocktail (Sigma)
NE-PER^TM Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, 78833)

dependent variables

Protein expression levels of beta Actin, gamma H2A.X, PARP-1, Cdc2 p34, BRCA1, Phospho-cdc2 (Tyr15), Phospho-BRCA1 (Ser1497), Rad51, XRCC1, and Lamin B1

control variables

15 × l protein A Sepharose 4B beads (Sigma)
BCA protein assay (Pierce, Rockford, IL, United States)
Western Blot procedures as described previously (Ruiz et al., 2019)

positive controls

Not specified

negative controls

Not specified

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