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. 2013 Jan 3;288(7):5241–5256. doi: 10.1074/jbc.M112.404996

FIGURE 5.

FIGURE 5.

The GPR17 promoter is selectively activated by CM. A, shown is a schematic representation of the human (h), mouse (m), and rat (r) GPR17 gene. The region upstream to the coding sequence has been analyzed with the Genomatix suite. Small blocks (from 600 bp to 1 kb) have been identified as putative promoter regions. Five regions in human, two in mouse, and two in rat were found (in gray). Only the region 1 is highly conserved among the three species. The human sequence 1 (909 bp) has been cloned in a pGL4 vector for the reporter assay. B, results of dual reporter luciferase assays show the GPR17 promoter activity in Oli-neu cells after transfection with the reporter construct pGL4-h909 or the corresponding empty vector. This was followed by incubation in Sato medium (control), neurobasal medium plus B27 (+NB), or CM for 24–48 h. Data are from three independent experiments and represent the mean ± S.E. Statistical analysis was performed using a two-way analysis of variance (***, p < 0.0001). C, cell extracts (30 μg protein) from Oli-neu cells incubated with either medium alone (−) or CM or neurobasal medium plus B27 (+NB) for the selected time points were analyzed by Western blotting using antibodies against GPR17. D, cell extracts from COS-7 (30 μg) or Oli-neu cells incubated with either Sato medium alone (−) or supplemented with CM for the selected time points were analyzed by Western blotting using antibodies against FoxO1 or GPR17. E, Oli-neu cells transiently transfected with cDNAs encoding for GFP-FoxO1 (FoxO1) or FLAG-FoxO1ADA were cultured in Sato medium for 48, fixed, and then labeled with anti-Ct-GPR17 antibodies and anti-FLAG antibody followed by secondary antibodies. Cells were examined using an Axiovert 200M confocal system equipped with a spinning disc. Arrows indicate FoxO1-transfected cells. Note that wild type protein is accumulated mainly in cytoplasm, whereas the constitutively active form (FoxO1ADA) is accumulated in the nucleus. The images are representatives from three independent experiments. Bar = 20 μm.