Evaluation of protein phosphorylation during adipogenesis

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.