Anti mcm2 n 19

The following reagents were used: β-NAD⁺ (Sigma), ³²P-NAD⁺ (Perkin Elmer), interferon α (Peprotech), lipopolysaccharid (LPS) of E. coli (Sigma), 12-O-Tetradecanoylphorbol-13-acetate (PMA) (Sigma), protease inhibitor cocktail (PIC) (Sigma), H₂O₂ (Merck KGaA), Glutathione-sepharose (Sigma), TALON metal affinity resin (BD Bioscience), ADPr (Adenosine 5′ diphosphoribose sodium-salt, Sigma), anti-HA (3F10, Roche), anti-HA (Covance), anti-PAR (Trevigen), anti-α-Tubulin (Sigma), anti-ARTD10 (5H11)2 (link), anti-ARTD10 purified rabbit antibodies10 (link), anti-Actin (C4, MP Biomedicals), anti-FLAG (Sigma), anti-GFP (Rockland), anti-MCM2 (N-19, Santa Cruz), goat anti-rat IgG (H + L) secondary antibody Alexa Fluor 555 conjugate (Invitrogen), anti-rabbit-HRP (Jackson Immunoresearch), anti-mouse-HRP (Jackson Immunoresearch), anti-rat-HRP (Jackson Immunoresearch). Rabbit polyclonal, purified ARTD8-specific antibodies were generated against the peptide NLVSDKIPKAKDTQG (aa 1193–1207).

Total protein extracts and chromatin fractionation were performed as previously described (Poli et al, 2016 (link)). Proteins were resolved by SDS–PAGE and transferred with a Trans‐Blot (Bio‐Rad). After blocking, proteins were either probed with anti‐RNAPII CTD (Abcam 8WG16, ab817), anti‐Rpb1‐S5P (Clone 3E8, Merck, 04‐1572), anti‐Rpb1‐S2P (Abcam, ab5095), anti‐PK for Maf1‐3PK strains (Novus Biologicals, NB600‐381), anti‐Rad53 (clone 11G3G6, custom made by GenScript), anti‐Mcm2 (N‐19, Santa Cruz, sc‐9839), anti‐tubulin (Thermo Fisher Scientific, MA1‐80017), or anti‐actin (clone C4, Sigma‐Aldrich, MAB1501). Blots were scanned with an ImageQuant LAS4000 mini (GE Healthcare), and semi‐quantitative determination of protein level was performed using the ImageJ (Fiji) software using tubulin, actin, or Mcm2 for normalization.

Formalin-fixed, paraffin-embedded (FFPE) tissue was sliced (4-μm thickness), and the sections were placed on silane-coated slides. Post deparaffinization, heat-based antigen retrieval at 95° C for 20 min in citrate buffer (pH 6.2), endogenous peroxidase blockade using 3% hydrogen peroxide, and blocking with normal horse serum (ABC Kit; Vector Laboratories, Burlingame, CA, USA) were performed. The primary antibodies used were as follows: anti-MCM2 (BM28; targeted to the C-terminus); mouse monoclonal, clone no. 610701, 1:2,000 (BD Transduction Laboratories, Cambridge, MD, USA); anti-MCM2 (N-19; targeted to the N-terminus); goat polyclonal, clone no. sc-9839, 1:250 (Santa Cruz Biotechnology, Inc., Dallas, TX, USA); anti-Ki-67 (MIB-1), mouse monoclonal, clone no. M7240, 1:200 (DAKO, Tokyo, Japan); and anti-cleaved Caspase-3 (CC3) (Asp175), rabbit monoclonal, clone no. 5A1E, 1:250 (Cell Signaling Technology, Danvers, MA, USA). Specimens treated with primary antibodies were incubated overnight at 4° C. Primary antibodies were detected using the ABC Kit (Vector Laboratories) for MCM2 (BM28), Histofine Simple Stain™ (Nichirei Bioscience, Tokyo, Japan) for MCM2 (N-19) and CC3, and diaminobenzidine (DAB; Vector Laboratories). Counter staining was performed using hematoxylin. The positivity of each protein expression was calculated in 10 areas of high-power field randomly chosen.