Coexpression and release of secretory proteins and Venus via the 2A peptide bridge in cultured neurons. A. Top, amino acid residues of the 2A peptide from Thosea asigna virus (T2A). The ribosomal skipping site (lack of peptide bridge formation between G and P) is indicated by a black arrowhead. Four amino acids (red) were added to create a constant C-terminal environment among different constructs. Bottom, schematic diagram of rAAVs. The theoretical molecular weights for the expected proteins of the rAAV encoded polypeptides are indicated in kDa above the boxed open reading frames. B. Primary cortical neurons (PCN) were infected with rAAVs expressing v-BMP7, v-BMP2 and v-Noggin. Immunoblots revealed BMP2 and BMP7 precursors and mature secretory morphogenic proteins and some BMP7-2A-Venus and BMP2-2A-Venus full-length proteins in lysates (L). In PCN supernatants (S) only mature morphogenic proteins could be detected. Venus was visible in PCN lysates and not in the corresponding supernatants. Venus was detected by anti-GFP antibodies. Anti-beta-actin was used as loading control. C. Schematic drawing of rAAVs encoding secreted Venus (sVenus) and cytosolic Venus. D. Immunoblots of PCN expressing rAAV-Venus (v-Venus) and rAAV-sVenus (v-sVenus). The v-Venus was not present in PCN supernatants, whereas sVenus was observed in lysates and supernatants using anti-GFP antibodies. E. After infecting PCNs with the indicated rAAV constructs immunocytology using anti-2A and anti-GFP antibodies showed co-expression of cytosolic Venus (green) and nuclear excluded BMPs or Noggin (red). On the right the expression of v-sVenus and v-Venus is depicted. Cell nuclei are stained with DAPI (blue) indicating the high efficiency of rAAV infection. Scale bar, 20 μm.