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Abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, IIe; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
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Subconfluent Rat-IR or NIH 3T3 cells were transiently transfected with PPARγ2 expression vector (SV-sport-PPARγ2) as described (8, 10). Twenty-four hours after transfection, cells were washed and fed with Dulbecco's minimal essential medium (DMEM) supplemented with 0.5% bovine serum albumin. Twenty-four hours later, cells were stimulated with EGF (20 ng/ml), TPA (100 ng/ml), TNF (50 ng/ml), insulin (5 μg/ml), or 30% serum in DMEM for 30 min. Untreated and treated cells were harvested into RIPA lysis buffer (24) supplemented with sodium vanadate (5 mM), NaF (100 mM), phenylmethylsulfonyl fluoride (2 mM), aprotinin (5 μg/ml), pepstatin (5 μg/ml), and leupeptin (5 μg/ml). Soluble proteins were separated by SDS-PAGE (10% gel, acrylamide:bis-acrylamide ratio of 100 with 5 M urea). Protein immunoblots were performed as described (25).
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54
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Rat-IR cells were transfected with expression vector for PPARγ and cell lysate harvested as described (26). After RIPA lysis, protein (200 μg) from each time point was dialyzed in buffer (50 mM tris, pH 8.5, 0.1 mM EDTA) in a microdialyzer (Pierce), and half of the dialyzed lysates were subsequently treated with calf intestine alkaline phosphatase (50 units) for 1 hour at 37°C.
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We thank members of the B.M.S. laboratory, especially P. Peraldi and R. Brun, for helpful discussions and suggestions. We also thank J. Blenis, T. Roberts, and C. Marshall for reagents. E.H. is supported by a postdoctoral fellowship from Sandoz-Dana-Farber drug discovery program. This work is supported by a NIH grant to B.M.S. (R37DK31405).
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