Ab32420

Cells were first lysed (Solarbio, Beijing, China) and then denatured in SDS buffer to obtain total protein. The protein was separated on a 10% SDS-polyacrylamide gel (30 mg per lane) and transferred to a PVDF membrane (Merck Millipore, MA, USA). After blocking in 5% skim milk for 1 h, the membrane was incubated with primary antibody overnight at 4 °C and then incubated with the secondary antibody (1:15,000, GE Healthcare, Cambridge, UK) for 1 h at room temperature. Then, visualization was performed with ECL chemiluminescence reagent (Merck Millipore, MA, USA).
The antibodies used in this study were as follows: CRABP2 (1:1,000, ProteinTech, Cat. # 10225-1-AP), BAX (1:1,000, Abcam, ab32503), BCL-2 (1:1,000, Abcam, ab32124), cleaved caspase-3 (1:1,000, Abcam, ab32042), PARKIN (1:1,000, ProteinTech, Cat. # 14060-1-AP), ATR (1:1,000, Abcam, ab2905), p-ATR (1:1,000, Abcam, ab178407), ATM (1:1,000, Abcam, ab32420), p-ATM (1:1,000, Abcam, ab81292), P53 (1:1,000, Abcam, ab32389), p-P53 (1:1,000, Abcam, ab33889), TET1 (1:1,000, Abcam, ab272900), DNMT3A (1:1,000, Abcam, ab188470), DNMT3B (1:1,000, Abcam, ab2851), P62 (1:1,000, ProteinTech, Cat. # 18420-1-AP), TOMM40 (1:1,000, Abcam, ab185543), α-tubulin (1:5,000, Abcam, ab7291), β-actin (1:5,000, ProteinTech, Cat. # 66009-1-Ig), GAPDH (1:20,000, ProteinTech, Cat. # 60004-1-Ig).

The antibody against BHRF1 was a gift from Jaap Middeldorp (VU University Medical Centre, The Netherlands). Antibodies against BZLF1 and BRLF1 were purchased from Argene (bioMérieux SA, Marcy l'Etoile, France). The rabbit monoclonal antibodies against phospho‐ATM (ab81292), ATM (ab32420), and the anti‐HA tag (1:5000 dilution; ab9110) rabbit polyclonal antibody were purchased from Abcam. The anti‐γ‐H2AX^ser139 antibody was purchased from EMD Millipore (Quincy, MA, USA). The antibodies against CHK2 (#3440) and phospho‐CHK2 (#2197) were purchased from Cell Signaling Technology (Danvers, MA, USA). All of the AlexaFluor‐conjugated and HRP‐conjugated secondary antibodies were purchased from Molecular Probes (New York, NY, USA). The HRP‐conjugated secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Western blot analysis was performed as previously described 24 and all the primary and the secondary antibodies used is 1:1000 and 1:5000 in dilutions unless otherwise specified. The signal intensity was measured by ImageJ software (http://rsb.info.nih.gov/ij).

PC tissue samples were fixed in 10% formalin (FUJIFILM) and embedded in paraffin. Staining was performed on 4 μm-thick sections, which were incubated at 4 °C overnight with rabbit anti-ATM (1:100; Cat. No. ab32420, Abcam) or rabbit anti-phospho S1981-ATM (1:100; Cat. No. ab81292, Abcam) as the primary antibody. In non-neoplastic cells, ATM expression was observed in islets and lymphoid cells, whereas phospho-ATM expression was observed in nerve cells, both of which served as internal controls. As a negative control, normal rabbit IgG (Cat. No. IS600, Dako, CA, USA) was used instead of the primary antibodies. ATM and phospho-ATM expression in cancer cells was evaluated visually by two pathologists (KH and TF) and classified into three grades according to the intensity of staining: score 0, null expression; score 1, faint expression; score 2, evident expression. A faint expression (score 1) was considered when the staining intensity was lower than that in adjacent internal controls (normal islet cells or nerve cells) on the same section, whereas an evident expression (score 2) was considered when the staining intensity was higher than or equal to that in the internal controls. Low ATM/phospho-ATM expression was assigned a score of 0, whereas high ATM/phospho-ATM expression was assigned scores of 1 and 2.

Protein extracts were obtained at 3, 5 and 7 days of no treatment or treatment with 50 μg/ml CDV. Whole cell lysates were prepared in Ripa buffer (Thermo Scientific, Brussels, Belgium) containing protease (Complete Mini, EDTA-free, Sigma-Aldrich, Diegem, Belgium) and phosphatase inhibitors (Active Motif, La Hulpe, Belgium). Protein concentrations were measured by the BCA assay (Thermo Scientific). Separation of proteins was performed with SDS-PAGE. Western Blot analysis was performed with anti-phospho-ATM (Ser1981) clone 10H11.E12 (Millipore, Billerica, MA, USA) and anti-ATM antibody (ab32420, Abcam, Cambridge, UK). Relative quantification was performed using actin (ab3280, Abcam) as a loading control. For each blot, the sum of intensity values was calculated and used for normalization of the values per blot.

Mice brains were gained to fix in 4% paraformaldehyde and dehydration with graded sucrose. After that, the brain was cut into 20 μm through a cryostat microtome (Leica, Wetzlar, Germany). Brain slices or primary microglia were moistened in 3 times with 1 × PBS and permeabilized with 0.25% Triton X‐100 for 20 min, and then blocked with 2% BSA (4240GR250, BioFroxx) for 2 h and incubated with primary antibody: anti‐Iba1 (ab5076, Abcam), anti‐P65 (8242, Cell Signaling Technology), or anti‐ATM (ab32420, Abcam) overnight at 4°C. Subsequently, microglia or slices were immersed in secondary antibody: Alexa 488 goat anti‐Iba1, Alexa 594 rabbit anti‐P65 or Alexa 647 rabbit anti‐ATM (A32814/A32754/ A32795, Invitrogen) for 1–2 h at 37°C. The nuclear was stained with DAPI (BD5010, Bioworld Biotechnology) for 10–15 min. Images were obtained by Olympus BX51 (Japan) fluorescence microscope and analyzed with ImageJ (ImageJ 1.5, NIH).

Total proteins were extracted using Complete Lysis buffer containing proteases inhibitors (04719956001 Roche Diagnostics, Mannheim, Germany) and phosphatase inhibitors (04906845001 Roche). Protein lysates were separated by NuPage 4–12% Bis-Tris Gel (NP0335BOX Invitrogen) under reducing conditions. Western blotting (WB) was carried out according to standard techniques, with rabbit anti-pATM (phospho S1981) ab81292, anti-ATM ab32420, and anti-b-tubulin (HRP) ab21058 (Abcam, Cambridge, MA, USA). Immunoreactive proteins were detected by ECL Prime (GE Healthcare, RPN2232) and a chemiluminescence gel documentation and analysis system (MINI HD, UVITEC, Cambridge, UK).

Antibodies used in this study are listed as follows: DNA-PKcs (ab32566, Abcam, UK, 1:5000), phosphorylated DNA-PKcs (ab124918, Abcam, 1:5000), phosphorylated ATM (ab81292, Abcam, 1:5000), ATM (ab32420, Abcam, 1:5000), PI3KCA (ab124918, Abcam, 1:1000), phosphorylated histone H2AX (2577, Cell Signaling Technology, USA, 1:500 for IB, 1:100 for IHC), H2AX (7631, Cell Signaling Technology, 1:1000), IRF9 (76684, Cell Signaling Technology, 1:1000), eIF-2α (5324, Cell Signaling Technology, 1:1000), phosphorylated eIF-2α (3398, Cell Signaling Technology, 1:1000), JNK (9252, Cell Signaling Technology, 1:1000), phosphorylated JNK (9255, Cell Signaling Technology, 1:1000), PERK(3179, Cell Signaling Technology, 1:1000), CHOP (2895, Cell Signaling Technology, 1:1000), Ki-67 (9449 s, Cell Signaling Technology, 1:400), cleaved-Caspase-3 (9664 s, Cell Signaling Technology, 1:1000 for IB, 1:500 for IHC), Caspase-3 (9662, Cell Signaling Technology, 1:1000), CD4 (25229, Cell Signaling Technology, 1:200), CD8 (98941, Cell Signaling Technology, 1:400), GAPDH (AP0063, Bioworld, USA, 1:10000), α-tubulin (ARG65693, Arigo Biolaboratories, China, 1:5000), β-tubulin (AP0064, Bioworld, 1:10000), M1 E1 and NS3 (produced by Beijing Protein Innovation, China, 1:2000). Anticancer compounds used in this study were purchased from Selleckchem.