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note
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9 cells were lysed in buffer containing 50 mM Hepes (pH 7.8), 500 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, 3 mM dithiothreitol, and 0.5 mM phenylmethylsulfonyl fluoride. Lysates were then applied to anti-Flag M2 agarose (Sigma) (30 μl of anti-Flag agarose per milliliter of lysate). After extensive washing, Smad4C and its associated proteins were eluted with Flag peptide (0.4 mg/ml) (12). The proteins were resolved on a 10% low-Bis polyacrylamide gel, transferred to nitrocellulose membrane, and digested with Lys-C. Five peptides from the 80-kD protein were sequenced and showed a perfect match to c-SnoN: KIILEEMK (amino acid residues 345 to 452 in SnoN), KTDAPSGMELQS (residues 366 to 377), KTVSYPD-VSLEE (residues 449 to 460), KVGIGLVAAASS (residues 503 to 514), and KLEMMIK (residues 663 to 669). Abbreviations for the amino acid residues are as follows: A, Ala; D, Asp; E, Glu; G, Gly; I, Ile; K, Lys; L, Leu; M, Met; P, Pro; Q, Gln; S, Ser; T, Thr; V, Val; and Y, Tyr.
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Y. Li, C. M. Turck, J. K. Teumer, E. Stavnezer, J. Virol. 57, 1065 (1986); E. Stavnezer, A. E. Barkas, L. A. Brennan, D. Brodeur, Y. Li, ibid., p. 1073.
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35S-express (0.25 mCi/ml, NEN) and chased for various periods of time as indicated in Fig. 3B. Cells were then lysed and SnoN isolated by immunoprecipitation.
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0345423762
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7 Ba/F3 cells were cocultivated with the transfected Bing cells for 24 hours, and the infected cells were selected by cell sorting on the basis of GFP expression.
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R. Dahl, M. Kieslinger, H. Beug, M. J. Hayman, Proc. Natl. Acad. Sci. U.S.A. 95, 11187 (1998).
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Two polyclonal antibodies to SnoN were raised, one against a glutathione-S-transferase (GST) fusion protein containing residues 1 to 366 of the human SnoN protein [antibody (Ab) 1330], and the other against a peptide located at the COOH-terminus of human SnoN (KELKLQILKSSKTAKE). For immunoprecipitation of endogenous SnoN, the peptide antibody was covalently coupled to protein A Sepharose and incubated with lysates from Hep3B cells stimulated with or without TGF-β1. SnoN bound to the antibody column was then eluted with an excess amount of immunizing peptide and analyzed by protein immunoblotting with anti-Smad4 (Santa Cruz) or with anti-SnoN (Ab 1330).
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We thank S. Pearson-White for providing the c-snoN cDNA, P. Kaufman for the human CAC1 probe, and H. Nolla for help with the FACS analysis. Supported by U.S. Department of Energy (DOE)-LBNL grant DE-AC03-76SF00098, DOE/OBER grant DE-AC03-76SF00099, Wendy Will Case Cancer Fund, California breast cancer research program award, and March of Dimes research grant to K.L S.L.S. was supported by a predoctoral fellowship from the National Science Foundation.
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