Anti lamin a c 636

Cell culture reagents were purchased from Invitrogen and Sigma. Compounds 25 and 26 (ERK5-IN-1) were originally provided by Professor Nathanael Gray and Dr. Jinhua Wang, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, USA then latterly compound 26 was purchased from Selleckchem. AX15836 and JQ1 are from Tocris. BIX02189 is from Selleckchem. Anti-HA antibody for immunoblots was provided by the Babraham Institute Monoclonal Facility^{40 (link),41 (link)} and anti-HA (12CA5) antibody for immuno-fluorescence^{68 (link)} was from Roche/Sigma. Phospho-ERK5 TEY (3371) antibody was from Cell Signaling Technology^{40 (link),41 (link)}. Phospho-T733 and phospho-S754 ERK5 antibodies were a kind gift from Dr. Atanasio Pandiella, University of Salamanca, Spain^{16 (link)}. Anti-Flag antibody (M2) is from Sigma^{69 (link)}. Anti-MEK5 antibody (AB3184) is from EMD Millipore^{22 (link)}, anti-lamin A/C (636)^{70 (link)}, ERK5 (C-7)^{26 (link)} and myc (9E10)^{71 (link)} antibodies are from Santa Cruz and anti-MEK1/2^{72 (link)}, phospho-T359 RSK^{73 (link)} and total RSK^{74 (link)} from Cell Signaling Technology. Secondary antibodies were from BioRad^{40 (link),41 (link)}.

Protein was prepared with ice-cold 1× RIPA Lysis and Extraction Buffer (89900; Thermo Fisher Scientific) containing protease inhibitor cocktail (11836153001; Roche). After incubation for 15 min on ice, lysates were clarified by centrifugation at 16,000g for 10 min at 4 °C. Protein concentration was assessed using Pierce protein BCA assay kit (23225; Thermo Scientific), and 20 μg of proteins were loaded for SDS-PAGE, then transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were blocked for 3 h at room temperature in TBST (0.05% Tween-20) supplemented with 5% non-fat milk. Anti-OGG1 (PA5-86046; Invitrogen), anti-MTH1 (PA5-52963, Thermo Fisher Scientific), anti-NEIL2 (PA5-78662; Invitrogen), anti-p65 (F-6) (sc-8008; Santa Cruz), anti-phospho-IRF-3 (Ser396) (4D4G) (#4947; cell signaling), anti-IRF-7 (G-8) (sc-74472, Santa Cruz) and anti-pS276 p65 (ab30623, Abcam) were used as primary antibody incubated overnight at 4 °C. β-actin (4970S; Cell Signaling Technology) and anti-Lamin A/C (636) (sc-7292, Santa Cruz) were used as internal loading control. Images were visualized by Amersham 680 imaging system.