Figure 1: PEDF expression (western blot; upper panel, Q-PCR; lower panel) in 3T3-L1 adipocytes during differentiation from fibroblasts into mature adipocytes (A) and the effects of energy deprivation (B). Differentiation was induced two days after the 3T3-L1 fibroblasts had reached confluence by treating them with the indicated medium for 48 h (day 0). Cells were re-fed with FCS-containing DMEM for the following 8 days (days 2-10). To study the effects of energy deprivation, FCS-containing medium was changed to DMEM with low glucose (1000 mg/L) on day 4. At the indicated time, cell lysates were lysed and centrifuged at 14,000 x g for 10 min at 4°C. After the resulting supernatants, including tissue protein extracts, had been incubated with anti- PEDF polyclonal antibody, protein A-Sepharose beads were used to precipitate immune complexes. After washing the beads, the immunoprecipitated proteins were subjected to SDS-PAGE, followed by electrophoretic transfer to a nitrocellulose membrane. Membranes were incubated for 1 hr at RT with anti- PEDF monoclonal antibody. After blotting with the indicated secondary antibody, detection was performed using an ECL chemiluminescent kit (upper panel). Total RNA from 3T3-L1 adipocytes was isolated with Isogen and cDNA was synthesized from purified RNA using a reverse transcriptase kit. For Q-PCR of PEDF (lower panel), we conducted real-time PCR using an ABI PRISM Model 7000. Representative data from four independent experiments are presented. *Significant difference (P <0.05) relative to PEDF expression in control cells.