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Volumn 274, Issue 5295, 1996, Pages 2100-2103

Inhibition of adipogenesis through MAP kinase-mediated phosphorylation of PPARγ

Author keywords

[No Author keywords available]

Indexed keywords

MITOGEN ACTIVATED PROTEIN KINASE;

EID: 0038776380     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.274.5295.2100     Document Type: Article
Times cited : (955)

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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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    • 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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