2A-released v-BMPs and v-Noggin are biologically active. A. BMP7 in lysates (L) and culture medium (S) of rAAV infected PCNs at different time points. Inset: Anti-BMP7 immunoblots of L (4 μl) and S (12 μl) used for quantification. B. Time course of v-BMP2 expression in PCN determined by ELISA of 4 μl L and 12 μl S. Inset: Noggin immunoblot of L and S of rAAV-Noggin-2A-Venus infected PCN at DPI 6. Precursor and released peptides are indicated by arrowheads; cystines containing forms by open arrows. C. Anti pSmad immunoblots of PCN (top) and HeLa cells (bottom) treated for 4 h with recombinant proteins or conditioned medium from rAAV infected PCNs as indicated. Ctrl.: non-infected PCN. Loading control: β-actin. D. left: H2O2 induced death of PCN as shown by lactate dehydrogenase (LDH) release 24 h after adding H2O2. Values are means ± SEM, One-way ANOVA, p < 0.0001. *p < 0.05, ***p < 0.0001 in comparison to untreated groups. D, E. Pretreatment with r-BMP7 (1.25 nM) and rAAV-mediated v-BMP7 expression reduced H2O2 induced LDH release in PCN. R-BMP7 was added 30 min before H2O2. One-way ANOVA, p < 0.0006. *p < 0.05 (Tukey’s posthoc test). LDH is given relative to controls (Ctrl.). F, V-BMP2 specific induction of the BMP responder GFP in BRE:GFP mice. RGB overlay fluorescent images of hippocampal DG region in coronal brain sections of (left) BRE:GFP mice injected with rAAV-BMP2-2A-tdTomato and (right) with PBS as control. GFP was visualized by anti-GFP (green), and cells nuclei with DAPI (blue). tdTomato auto-fluorescence is in red. Hilus region (hi); molecular layer (ml) of the dentate gyrus (DG); mossy fiber projections (mf). Arrows indicate GFP expressing cells in the granular cell layer (gr). Blood vessels show an unspecific fluorescence in both brain sections (stars). Scale bar; 0.5 mm.
Heinonen et al. BMC Neuroscience 2014 15:38 doi:10.1186/1471-2202-15-38