Anti mre11

For preparation of protein extracts, cells were lysed in cell extraction buffer (50 mM Tris pH 7,4, 150 mM NaCl, 2 mM EGTA, 2 mM EDTA, 25 mM NaF, 0.1 mM Na₃VO₄, 0.1 mM PMSF, 2 mg/ml Leupeptin, 2 mg/ml Aprotinin, 0.2% Triton X-100, 0.3% NonidetP-40) for 30 min on ice. Protein extracts were cleared through centrifugation at 16100 rcf for 15 min, and supernatants were separated through SDS-PAGE and transferred to nitrocellulose membranes. Antibodies to NBN, SMC1-pS966, KAP1-pS824 were obtained from Novus Biologicals (rabbit polyclonal); anti CHEK2-pS19 from Cell Signaling (rabbit polyclonal); anti RAD50 (mouse monoclonal) from Abcam; anti MRE11 (mouse monoclonal 12D7) from GeneTex; PARP1 and cleaved PARP1 (rabbit polyclonal) from Cell Signaling, and anti β-Actin (mouse monoclonal) from Sigma. Anti-mouse and anti-rabbit horseradish peroxidases labelled secondary antibodies were purchased from GE Healthcare. Visualization of immunoreactive bands was performed by using ECL (Thermo Scientific/Pierce), and their intensity was determined using ImageJ quantitation software.

Western blots were performed with primary antibodies anti-RAD50 (GTX70228, Genetex, Irvine, CA, USA), anti-MRE11 (GTX70212, Genetex, Irvine, CA, USA), anti-NBS1 (GTX70224, Genetex, Irvine, CA, USA), anti-6xHIS (#12698, Cell Signaling Technology, Danvers, MA, USA), anti-α-tubulin (GTX112141, Genetex, Irvine, CA, USA), and anti-P84 (GTX70220, Genetex, Irvine, CA, USA), as described previously [36 (link),37 (link)].

Whole cell and nuclear extracts were prepared as described previously^{63 (link)}. Western blot analysis was performed as described^{64 (link)} with the following primary antibodies: anti-TRF1 (#5745, 0.5 μg/ml)^{63 (link)}, anti-TRF2 (4A794, 2.5 μg/ml, Novus Biologicals, Littleton, CO, USA), anti-POT1 (#978, 1:1,000, provided by Dr. Titia de Lange), anti-TIN2 (1:1,500, provided by Dr. Zhou Songyang), anti-TPP1 (#467, 1:1,000, provided by Zhou Songyang), anti-RAP1 (1:1,000, provided by Zhou Songyang), anti-RIF1 (#1060, 1:1,000, provided by Titia de Lange), anti-dyskerin (H-300, 2 μg/ml, Santa Cruz Biotechnology, Dallas, TX, USA), anti-MRE11 (12D7, 1:500, GeneTex, Irvine, CA, USA), anti-RAD50 (13B3, 1:1,000, GeneTex), anti-NBS1 (#3002, 1:1,000, Cell Signaling Technology, Danvers, MA, USA), anti-BLM (ab476, 1:2,000, Abcam, Cambridge, UK), anti-WRN (ab200, 1:2,000, Abcam), anti-ATM (2C1, 2 μg/ml, Santa Cruz Biotechnology), anti-PARP-1 (C2-10, 1:2,000, Pharmingen, Franklin Lakes, NJ, USA), anti-tankyrase-1 (H-350, 2 μg/ml, Santa Cruz Biotechnology), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; RDI-TRK5G4-6C5, 0.5 μg/ml, Research Diagnostics, Flanders, NJ, USA), anti-α-tubulin (B-5-1-2, 1:1,000, Sigma), anti-lamin A/C (636, 2 μg/ml, Santa Cruz Biotechnology) and anti-hTERT (1531-1/Y182, 1:1,000, Abcam). Images were processed with Photoshop CS5 (Adobe).

Cells were fixed (10min, 22°C) in 4% paraformaldehyde/PBS (made from 16%(w/v) formaldehyde; TAAB, UK), followed by permeabilization (10min, 22°C) in Triton buffer (0.5% Triton X-100, 20mM Hepes KOH pH7.9, 50mM NaCl, 3mM MgCl2, 300mM Sucrose). Cells were blocked in 0.05% Tween-20/3% BSA/PBS (15min, 22°C) and incubated (overnight, 4°C) in primary antibodies diluted in blocking buffer. Antibodies used: mouse monoclonal anti-Ad-DBP (37.3) (a gift from K. Benihoud, Institut Gustave Roussy, Villejuif, France), rabbit polyclonal anti-phospho-histoneH2A.X (Ser139) (Cell Signalling Technology, MA), mouse monoclonal anti-E1A and/or rabbit polyclonal anti-Mre11 (Genetex Inc., CA). Coverslips were washed twice in blocking buffer and incubated (1h, 22°C) in secondary antibodies (goat anti-mouse or anti-rabbit AlexaFluor^® 594 and/or goat anti-mouse AlexaFluor^® 488) diluted in blocking buffer.

Primary antibodies used in this study were as follows (Table S1): Anti-RecQL4 antibody was prepared by Abfrontier by immunizing rabbits with recombinant N-terminal (amino acid residues 1–241) RecQL4. Other antibodies used in this study were as follows: Anti-pATM (Ser-1981, #4526, Cell Signaling), anti-Mre11 (GTX30294, GeneTex), anti-Nbs1 (A7703, ABclonal), anti-Rad50 (sc-74460, Santa Cruz), anti-Rad51 (GTX100469, GeneTex), anti-RPA32 (MABE-286, EMD Millipore), anti-Usp28 (A9292, ABclonal), anti-Skp2 (sc-7164, Santa Cruz), anti-γH2AX (A300-081A, Bethyl and 05-636, EMD Millipore), anti-lamin B1 (ab16048, Abcam), anti-GAPDH (sc25778, Santa Cruz), anti-FLAG M2 (Sigma), and anti-HA (AE008, ABclonal). The following antibodies were used as secondary antibodies in immunofluorescence microscopy: Alexa Fluor 488 anti-mouse IgG (A11001, Thermo Fisher), Alexa Fluor 488 anti-rabbit IgG (A11008, Thermo Fisher), Alexa Fluor 594 anti-mouse IgG (A11005, Thermo Fisher), and Alexa Fluor 594 anti-rabbit IgG (A11012, Thermo Fisher).