FIGURE 3.

NaPB increases the expression of BDNF and NT-3 in primary mouse astrocytes via CREB. A, cells were treated with different doses of NaPB for 2 h followed by monitoring mRNA expression of CREB by qPCR (top) and RT-PCR (bottom). B, time course (30, 60, 120, 180, and 360 min; 0.2 m m ) responses of CREB mRNA transcription was monitored by qPCR (top) and RT-PCR (bottom). C, total CREB expression is significantly up-regulated by NaPB. Cells stimulated with 0.05, 0.1, 0.2, or 0.5 m m NaPB for 24 h were subjected to immunoblotting with anti-CREB and anti-β-actin and analyzed by densitometry (top). D, NaPB time-dependently induces significant increases in CREB phosphorylation. Cells stimulated with 0.2 m m NaPB for 15, 30, 60, or 120 min were subjected to immunoblotting with anti-CREB-Ser(P)133 (pCREB) anti-CREB, and anti-β-actin and analyzed by densitometry (top). E, NaPB-mediated induction of phosphorylated CREB is localized to the nucleus. Astrocytes stimulated with 0.2 m m NaPB for 60 min were immunostained with anti-GFAP (green) and anti-CREB-Ser(P)133 (red) and treated with DAPI (blue). Whole cells and nuclei were captured using ×40 and ×60 objective lenses, respectively. Scale bar, 2 μm. F, the phosphorylation state of CREB is elevated by NaPB. Cells seeded in 96-well plates were treated with 0.2 m m NaPB for 15, 30, 60, or 120 min, fixed, permeabilized, subjected to infrared in-cell Western blotting with anti-CREB (blue) and CREB-Ser(P)133 (orange) antibodies, and analyzed for relative fluorescent units (top). G, NaPB induces DNA binding of CREB. Cells stimulated with 0.2 m m NaPB for 15, 30, or 60 min (left) were subjected to EMSAs. A supershift assay was performed with anti-CREB antibodies or IgG (right panel) to verify the presence of CREB in complex. H, NaPB elevates transcriptional activity of CREB. Astrocytes were transfected with pCRE-Luc (blue), pAP-1-Luc (orange), and pNFκB-Luc (navy). After 24 h of transfection, cells were treated with NaPB for 4 h followed by monitoring the luciferase activity in cell extracts. I and J, CREB siRNA successfully suppresses CREB expression. Cells were unstimulated or transfected with 0.25 μg of scrambled or CREB siRNA for 48 h and subjected to immunoblotting (I) with anti-CREB (blue) and anti-CREB-Ser(P)133 (orange) antibodies followed by densitometry (J). K and L, NaPB-mediated induction of BDNF and NT-3 mRNA is dependent upon CREB. Cells transfected with 0.25 μg of control or CREB siRNA for 48 h were unstimulated (blue) or treated with 0.2 m m NaPB (orange) or 0.2 m m NaFO (navy) for 6 h, and BDNF (K) and NT-3 (L) mRNA was examined by qPCR. M–O, NaPB-mediated induction of BDNF and NT-3 protein is dependent upon CREB. Cells transfected with 0.25 μg of control or CREB siRNA for 48 h were unstimulated (blue) or treated with 0.2 m m NaPB (orange) or 0.2 m m NaFO (navy) for 24 h, lysed, and immunoblotted with anti-BDNF and anti-NT-3 antibodies (M). Densitometry was performed on blots for NT-3 (N) and BDNF (O). Results represent three independent experiments. Each treatment condition is represented by two independent bands in C, D, I, and M. Data were analyzed for statistical significance by one-way ANOVA (*, p < 0.05; **, p < 0.01). Ab, antibody; MRGD, merged image; N.S., nonspecific; RLU, relative luciferase units; Scr, scrambled. Error bars, S.D.