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. 2015 Nov 11;10(11):e0142067. doi: 10.1371/journal.pone.0142067

Fig 5. Validation of expression, secretion, and activity of viral vector-derived BDNF.

Fig 5

(A) The rAAV2/1-CMV-eGFP vector almost exclusively induces eGFP expression in neurons (NeuN) and not in glial cells (GFAP). Filled arrowheads point towards double-labeled cells. Scale bars: 20 μm. (B) Unilateral rAAV2/1-CMV-BDNF vector-induced BDNF upregulation is visualized through IHC. Scale bar: 200 μm. (C) Total BDNF levels in BDNF vector-transduced SC are sharply enhanced compared to non-transduced SC in the same animals (internal control) and to both SCi of eGFP vector-transduced mice, quantified via ELISA. (D) Western blot analyses show an upregulation of mBDNF levels in BDNF vector-transduced SC compared to internal controls and to eGFP vector- transduced animals. (E) In contrast to HEK293T cells transduced with the eGFP control vector, HEK293T cells transduced with BDNF vector secrete large amounts of BDNF in the culture medium, as quantified by ELISA on medium harvested 24 h and 72 h post-transduction. (F) Retinal explants cultured in medium supplemented with recombinant BDNF (rBDNF) or vector-derived BDNF (vBDNF) show enhanced neurite outgrowth areas compared to control explants (eGFP). N = number of explants, derived from three mice. Key: BDNF, brain-derived neurotrophic factor; mBDNF, mature form of BDNF; eGFP, enhanced green fluorescent protein; DAPI, 4’,6-diamidino-2-phenylindole; SC, superior colliculus; rAAV2/1, recombinant adeno-associated viral vector serotype 2/1; CMV, cytomegalovirus promoter; IHC, immunohistochemistry; ELISA, enzyme-linked immunosorbent assay; ONC, optic nerve crush; OHT, ocular hypertension; dpi, days post-injury; HEK293T, Human embryonic kidney cell line.