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Fig. 3. NMDA stimulates the release of VEGF from proliferating hippocampal NPCs. (A) Real-time PCR analysis of BDNF, PDGF and VEGF mRNAs 24 hours and 72 hours after a single 24-hour pulse of each substance. The expression of each gene was normalized to the amount of GAPDH to obtain the relative level of its transcript (n=5 per group) and is shown relative to the expression levels in control cells at 24 hours. (B) Western blot of VEGF. Secretion of VEGF into the culture medium 72 hours after the single 24-hour pulse of each treatment. VEGF (10 ng/ml) was loaded as a positive control. (C) Addition of VEGF or BDNF increases the fraction of BrdU+ (red). VEGF (50 ng/ml) or BDNF (50 ng/ml) was added to the culture medium in the absence of bFGF for 72 hours, and proliferating cells were labeled by BrdU for 1 hour before fixation. All the cells were counterstained with DAPI (blue). The percentage of BrdU+ cells [BrdU(+)] cells is shown. Bar, 100 µm. (D) Quantitative RT-PCR analysis showing that Bcl2 and Bax mRNA levels were increased and decreased, respectively, after exogeneous VEGF application for 24 hours. Expression levels of Bcl2 and Bax were normalized to the amount of GAPDH expression to calculate the relative amount of gene transcript (n=3 per group). Expression levels at each concentration are depicted relative to the expression of the control 3 days after the onset of stimulation for comparison. MK, MK801. Error bars indicate ± s.e.m. CTL, control. *P<0.05, **P<0.01, **P<0.001.
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