Co-expression of BDNF and Venus using P2A peptide. (A) Protein expression with the Venus-P2A-BDNF construct, which was designed to produce Venus-P2A fusion protein and BDNF separately, was confirmed by performing an immunoblotting analysis for Venus, a GFP mutant, (A1) or BDNF (A2). In this experiment, the Venus-P2A-BDNF construct was transfected to HEK293T. Even though a weak band was noted at the position of the non-cleaved fusion protein for Venus-P2A-BDNF, almost all signals were detected for the cleaved Venus-P2A. (A2) In the culture medium of HEK293 cells transfected with the Venus-P2A-BDNF construct (lane a), a specific band was observed at the same molecular weight as that for the control from cells showing BDNF expression (lane b); this indicated that BDNF, when cleaved from the Venus-P2A-BDNF construct, followed the normal course of processing and secretion. (B) Schematic diagrams of the expression vectors. (C-F) Immunohistochemical staining for Venus (green) and BDNF (magenta) in the dentate gyrus (C, D) and CA3 region (E, F) of cultured slices transfected with the mock Venus-P2A- (C, E) or Venus-P2A-BDNF-carrying (D, F) virus. In D and F, Venus-positive cells showed intense BDNF staining in slices transfected with the Venus-P2A-BDNF virus, whereas endogenous BDNF and Venus were rarely colocalized in slices transfected with the mock virus (C and E). ML: molecular layer, GCL: granule cell layer, DH: dentate hilus, SL: stratum lucidum, SP: stratum pyramidale, and SO: stratum oriens.