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2 protocols using anti fen 1

1

Immunoblotting Characterization of DNA Replication Proteins

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Primary antibodies used were anti-PCNA (for western blotting, 1:500 dilution, clone PC10, sc-56, lot# E2418, Santa Cruz Biotechnology, Dallas, TX), anti-PCNA (for immune-depletion, clone F2, sc-25280, lot #H1417, Santa Cruz Biotechnology), anti-POLD1 (for western blotting, 1:1000 dilution, rabbit polyclonal, 15646-1-AP, Proteintech, Rosemont, IL), anti-FEN-1 (for western blotting, 1:1500 dilution, rabbit polyclonal, A300-256A, Bethyl Laboratories, Montgomery, TX), anti-LIG1 (for western blotting, 1:1000 dilution, clone 10H5, GTX70141, lot#809703999, GeneTex, Irvine, CA), and anti-RFC4 (for western blotting, 1:1000 dilution, clone 1320, GTX70285, lot#14130, GeneTex).
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2

Cellular Protein Expression Profiling

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Cell extracts were obtained by lysing cells for 15 min at 4C in buffer containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 0.4% NP-40, 10 mM MgCl2 and 5 mM DTT, supplemented with protease and phosphatase inhibitors (Halt Protease & Phosphatase Inhibitor Cocktail; ThermoFisher Scientific). After centrifugation (10,000 x g, 20 min), supernatants were diluted (v/v) in 50 mM Tris-HCl (pH 8.0), 0.4% NP-40 and 5 mM DTT. Proteins were separated by SDS-PAGE and immunoblotted with the following antibodies at dilutions recommended by the manufacturer: anti-pan-actin (MAB1501; Millipore), anti-ARTEMIS (#13381; Cell Signaling Technology), antimCherry (GTX128509; GeneTex), anti-CtIP (A300–488A; Bethyl), anti-FEN1 (A300–255A; Bethyl), anti-KAP1 (A300–274A; Bethyl), anti-DNA ligase3 (A301–636A; Bethyl), anti-MRE11 (A303–998A; Bethyl), anti-MUS81 (ab14387; Abcam), anti-PNKP (A300–257A; Bethyl), anti-SETX (A301–104A; Bethyl), anti-TDP1 (H00055775-A01; Abnova Corporation), anti-atubulin (T5168; Sigma-Aldrich), anti-XPF (A301–315A; Bethyl), anti-XPG (A301–484A; Bethyl), and anti-XRCC1 (A300–065A; Bethyl). Immunoblotting was revealed by chemiluminescence using a ChemiDoc MP Imaging System (Bio-Rad).
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