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. 2016 Jun 27;6:28648. doi: 10.1038/srep28648

Figure 4. Dexras1-induced activation of MAPK is dependent on Shc-Grb2 pathway.

Figure 4

(a) Immunoprecipitation and western blotting of Dexras1-FLAG and Shc-HA in 3T3-L1 cells. After stimulation of insulin (1 μg/ml) for 1 h, cell lysates were immunoprecipitated with anti- HA antibody and analyzed with anti-FLAG antibody. (b) Immunoprecipitation and western blotting of Dexras1-FLAG or H-ras-FLAG and Raf-HA. After stimulation of insulin (1 μg/ml) for 1 h, cell lysates were Immunoprecipitated with anti-HA antibody and then analyzed with anti-FLAG antibody. (c) 3T3-L1 cells were transfected with pcDNA3-Dexras1-FLAG or pcDNA3-H-ras-FLAG, and then differentiation was induced using the indicated hormone cocktails (MI, IBMX and insulin; MDI, IBMX, dexamethasone, and insulin). Oil-red-O staining was carried out on day 8. (d) 3T3-L1 cells were transfected with pcDNA3-Dexras1-FLAG or pcDNA3-H-ras-FLAG and incubated with insulin (ins) for 1 h. Cells were fixed and subjected to immunofluorescence analysis with antibody against FLAG (green). Nuclei were stained with DAPI and fluorescence was visualized by confocal microscopy. Scale bar = 50 μm. (e) 3T3-L1 cells were transfected with control, Shc, or Grb2 siRNAs, and differentiation was induced by MDI. Oil-red-O (ORO) staining was performed on day 8. ERK activation was evaluated by immunoblot at the indicated time points. (f) A chromatin immunoprecipitation study. The promoter region of Dexras1 was presented with putative GRE, oligonucleotides for PCR, and transcription start site (+1). Anti-GR antibody (αGR) was used for this assay. The resulting PCR products were resolved in 1.2% agarose gel. (g) A schematic model of Dexras1 functions in adipogenesis. Dexras1 is expressed by glucocorticoid and its receptor complex, remaining in the cytoplasm until insulin or IGF-1 is treated. When IGF-1R is activated by the hormone, Dexras1 translocates to the plasma membrane interacting with Shc, thereby transferring the IGF-1 signaling through Shc/Grb2, Raf, MEK, and MAPK. The Dexras1 is involved only in MAPK pathway, not interfering AKT/PKB signal. The activated MAPK then phosphorylates C/EBPβ protein, which mediates mitotic clonal expansion and further adipogenic program.