title: Evaluation of protein phosphorylation during adipogenesis.
abstract: Adipocyte differentiation is a complex process that involves the sequential expression of various adipocyte-specific genes controlled by signaling pathways and transcription factors for which phosphorylation plays a crucial regulatory role. CCAAT/enhancer-binding proteins and peroxisome proliferator-activated receptors are the most important transcriptional regulators in adipogenesis, and the functions of these proteins are regulated by various phosphorylation events. Because cultured 3T3-L1 preadipocytes are commonly used as a model for adipocyte differentiation, we used these cells for a proteomic analysis to identify kinases, phosphatases, and phosphosites that participate in adipogenesis. In addition to the phosphoproteomic analysis, we provide a detailed description of Western blotting, an in vitro phosphorylation assay, enzyme-linked immunosorbent assay, and phosphorylation site mutagenesis to fully characterize the phosphorylation of proteins and verify their roles in adipogenesis.
authors: Key Laboratory of Molecular Medicine, The Ministry of Education, Department of Biochemistry and Molecular Biology, Fudan University Shanghai Medical College, Shanghai, PR China.
authors: Xi
authors: X
authors: Li
authors: Key Laboratory of Systems Biology, Shanghai Institutes for Biological, Sciences, Chinese Academy of Sciences, Shanghai, PR China.
authors: Rong
authors: R
authors: Zeng
authors: Key Laboratory of Molecular Medicine, The Ministry of Education, Department of Biochemistry and Molecular Biology, Fudan University Shanghai Medical College, Shanghai, PR China. Electronic address: qqtang@shmu.edu.cn.
authors: Qi-Qun
authors: QQ
authors: Tang
mesh_terms: D041721:3T3-L1 Cells; D017667:Adipocytes / Q000737:chemistry / Q000166:cytology / Q000378:metabolism*; D050156:Adipogenesis; D000818:Animals; D015153:Blotting, Western / Q000379:methods; D022762:CCAAT-Enhancer-Binding Proteins / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D051379:Mice; D016297:Mutagenesis, Site-Directed / Q000379:methods; D047495:PPAR gamma / Q000378:metabolism; D010750:Phosphoproteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D022762:CCAAT-Enhancer-Binding Proteins; D047495:PPAR gamma; D010750:Phosphoproteins; D011494:Protein Kinases
keywords: Adipogenesis; Phosphoproteomic analysis; Phosphorylation; Phosphorylation site mutagenesis; Sandwich ELISA for phosphoprotein