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Figure S2. Purification of the MDC1–MRN complex from HeLa nuclear extract. H2AX phosphopeptide pull down was performed as described previously (Stucki, M., J.A. Clapperton, D. Mohammad, M.B. Yaffe, S.J. Smerdon, and S.P. Jackson. 2005. Cell. 123:1213–1226), but the beads were extensively washed with a high-stringency buffer (50 mM Tris-HCl, pH 8.0, 200 mM NaCl, and 0.5% NP-40). The bound proteins were eluted with SDS loading buffer and separated on an SDS-polyacrylamide gel followed by silver staining. The protein bands were identified by parallel immunoblot analysis using antibodies against human MDC1, RAD50, NBS1, and MRE11 (not depicted).