The Flag-tagged hTERT complex was obtained by co-IP with anti-Flag from 5 × 108 MCF7 cells stably expressing Flag-hTERT. Cells were lysed in high-salt IP buffer [20 mM tris-HCl at pH 8.0, 1 M NaCl, 1.5% Nonidet P-40 (NP40), 5 mM EDTA, and 5 mM β-mercaptoethanol supplemented with protease inhibitors] and immunoprecipitated with anti-Flag M2 agarose. After washing four times with the high stringent lysis buffer, the immunoprecipitants were resolved on gradient SDS–polyacrylamide gel electrophoresis (PAGE), silver-stained, and subjected to MS sequencing and data analysis. In-solution and in-gel digestion were performed according to a previously published method (48). Briefly, gel bands were minced and destained with 50% acetonitrile in 50 mM ammonium bicarbonate. Proteins were reduced with 10 mM dithiothreitol (DTT) at 56°C, followed by alkylation with 55 mM iodoacetamide at room temperature in the dark. Trypsin digestion was performed overnight at 37°C with gentle shaking. Peptides were extracted using 1% trifluoroacetic acid in 50% acetonitrile. Samples were vacuum-dried and reconstituted in 0.1% formic acid for subsequent MS analysis. The treated samples were examined by nano–LC-MS/MS (nanoACQUITY UPLC and SYNAPT G2 HD mass spectrometer, Waters). MS/MS data were obtained with data-dependent analysis mode and processed with PLGS 2.4 software (Waters), and the resulting peak list was searched against the National Center for Biotechnology Information database with the MASCOT search engine.

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