Neurotrophins promote revascularization by local recruitment of TrkB⁺ endothelial cells and systemic mobilization of hematopoietic progenitors
J. Clin. Invest. Pouneh Kermani, et al. 115:653 doi:10.1172/JCI22655 [Go to this article.]

Figure 2
Expression of biologically active BDNF protein after adenovirus-mediated gene delivery. (A) 293 cells were infected with AdBDNF or AdGFP at an MOI of 100, and media was harvested 48 hours after infection. Secretion of BDNF was confirmed by Western blot analysis after protein separation by SDS-PAGE. The 13-kDa immunoreactive band (arrow) is consistent with mature BDNF, and the 29-kDa and 17-kDa species correspond to incompletely processed proforms that are released when the gene is expressed at high levels. (B) The biological activity of BDNF expressed by adenovirus vector was evaluated using PC12 cells stably expressing TrkB. Media from 293 cells infected with AdBDNF, AdGFP, or rBDNF were added to cells, and neuritogenesis (the presence of neurite processes greater than one cell body in length) was assessed 24 hours after treatment using Normasky imaging; neurite outgrowths were visualized with a 20× objective. (C) In vivo expression of BDNF following injection of AdBDNF in the tail veins was measured by ELISA using a commercially available kit and a recombinant BDNF standard (BDNF E_max ImmunoAssay System). The BDNF E_max kit has a minimum sensitivity of 7.8 pg/ml. The ELISA for BDNF was carried out on plasma samples of SCID mice injected in the tail vein with 1 × 10⁹ PFU of AdBDNF. Blood was collected every 2–3 days after adenovirus vector administration. Absorbance was measured at 450 nm using a microplate reader and the BDNF concentration was normalized to that of recombinant protein.