Ab214

Cell lysates were prepared in Pierce IP lysis buffer (Thermo; 87788). Total protein concentration was determined by BCA assay and 500 μg lysate was incubated with anti-MRE11 (ab214; abcam, Cambridge, UK) at 4 °C overnight on a tube rotator and then added to protein A/G plus agarose (sc2003; Santa Cruz, Dallas, TX) for 1 h at 4 °C. The beads were washed five times in PBS-Tween 0.05% and binding proteins eluted by boiling (95 °C for 10 min) in 40 μL Laemmli buffer before SDS-PAGE gel running. Ten percent of the total lysate was retained as the load fraction. The antibodies used were anti-MRE11 (ab30725; abcam, Cambridge, UK), anti-RAD50 (3427; CST, Danvers, MA), anti-NBS1 (NB100-143; Novus Biological, Centennial, CO), anti-myc-tag (2278, CST, Danvers, MA). To isolate SPRTN-interacting proteins, whole cell lysates were prepared in lysis buffer (50 mM Tris-HCl pH7.4; 150 mM NaCl; 0.1% NP-40; 1 mM EDTA, and 5% glycerol; protease and phosphatase inhibitors) containing 500 U/ml of benzonase at 4 °C. Flag-tag protein complexes were separated using the anti-flag M2 antibody (F1804; Merck, Whitehouse Station, NJ) and washed five times in IP buffer containing 0.05% NP-40 and eluted in 3xFlag peptide (F4799; Merck) for 30 min at RT.

Rabbit antibodies against Tks5 (M-300), MAP4 (H-300), HA (Y-11) and mouse antibody against Grb2 (C-7) were purchased from Santa Cruz Biotechnology. Mouse antibody against Cortactin (clone 4F11) was purchased from Millipore. Antibodies against N-WASP (mouse, ab56454), NogoA/B (rabbit, ab47085), CD2AP (rabbit, ab205017), MMP14/MT1-MMP (rabbit, ab51074) and MRE11 (mouse, ab214) were from Abcam. Rabbit antibody against SHC1 (610081) and mouse antibodies against Nck (610099) and SHC1 (610879) were from BD Transduction Laboratories. Anti-α-Tubulin (mouse, clone DM1A) and anti-FGD1 (rabbit, HPA000911) were from Sigma Life Science. Biotinylated proteins were detected either by Alexa Fluor594 Streptavidin (405240, BioLegend) or Streptavidin-HRP (21130, Thermo Scientific).

U-2 OS^ATRX nuclear extracts were prepared using Cellytic Nuclear Extraction kit (Sigma) in the absence of detergent and equilibrated into IP buffer (20 mM HEPES pH 7.4, 1% Triton X-100, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA+protease inhibitor cocktail). Immunoprecipitations were performed overnight at 4 °C using 10 μg anti-RAD50 antibody (Abcam ab89) or 10 μg anti-MRE11 antibody (Abcam ab214) with protein G dynabeads. Beads were washed four times in IP buffer and bound proteins eluted into SDS loading buffer (Laemmli) by heating at 90 °C for 5 min. Western blotting was performed using anti-ATRX antibody (Santa Cruz sc-15408).

Protein lysis buffer was prepared in 50 mmol/L HEPES, 100 mmol/L NaCl, 10 mmol/L EDTA, 1% Triton X-100, 4 mmol/L Na pyrophosphate, 2 mmol/L sodium orthovanadate, 10 nmol/L NaF, and 50 mmol/L B-glycerophosphate. Cells were lysed in lysis buffer containing a cocktail of proteinase inhibitors (Roche, Mannheim, Germany). Protein quantification of the lysates was performed using a BCA protein assay (Thermo Fisher, Waltham, MA, USA) and 30μg of protein was resolved on 4% to 20% polyacrylamide gels and transferred onto nitrocellulose membranes. The resulting membranes were incubated with blocking buffer (Li-cor Biosciences, Lincoln, NE, USA) and primary antibodies. The antibodies used were mouse polyclonal anti-MRE11 (ab214; abcam, Cambridge, UK), rabbit anti-phospho-histone H2A.X (Ser139) (#2577; Cell Signalling Technology, Danvers, MA, USA), and mouse monoclonal anti-β-actin (ab6276; abcam, Cambridge, UK). Fluorochrome-conjugated secondary antibodies (Li-cor Biosciences, Lincoln, NE, USA) were used and detected by infrared scanning densitometry using the Li-cor Odyssey Infrared Detection System (Li-cor Biosciences, Lincoln, NE, USA).

SW620, DLD1, HCT-116 and HCT-116 chr3 cells were trypsinised, collected and lysed in Cell Panel Lysis Buffer with complete protease and phosphotase inhibitors. Proteins were separated by gel electrophoresis and transferred to nitrocellulose membrane by Western blot. Membranes were probed, at a concentration of 1:1000 unless stated otherwise, for ATM (sc-23921, Santa Cruz Biotechnology), MSH2 (556349, BD Pharmigen), MLH1 (WH0004292M2, Sigma Aldrich), MSH6 (610918, BD Transduction), MRE11A (ab214, Abcam), RAD50 (611010, BD Transduction), NBS1 (NB100-143, Novus Biologicals), FEN1 (ab109132, Abcam) and β-Actin (A5441, Sigma Aldrich).