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Figure S2. Additional fractionation of TRF1–TIN2 complexes from HeLa cell nuclear lysate and the efficiency of our nuclear extraction protocol. (a) HeLa cell nuclear extracts (5 ml) were fractionated on a Superdex S-200 HR column (GE Healthcare) as described in Materials and methods and analyzed for the indicated proteins by Western blotting. 20 µl aliquots of the indicated fractions were loaded. Each fraction after the column void volume (fraction 13) was collected and analyzed. Molecular sizes were derived from a standard curve based on the elution of standards described in Materials and methods. (b) Nuclear extraction efficiently liberates telomeric proteins. To assess the efficiency of our extraction protocol, exponentially growing HeLa cells (1L) were serially extracted, maintaining equivalent buffer/cell ratios for all steps. Nuclear extraction yielded a cytoplasmic fraction (lane 1), nuclear extract (lane2), and an insoluble pellet. The insoluble pellet was further extracted by sonication and DNAse I treatment (2 h at 25 °C) in nuclear extraction buffer supplemented with 5 mM CaCl and MgCl (lane 3). The remaining pellet was resuspended in SDS buffer, sonicated, and boiled for 10 min. Equivalent volumes of each fraction were analyzed by Western blotting for the indicated proteins.