Mca rppgfsafk dnp oh fluorogenic peptide substrate

After chemical lysis of the cells in lysis buffer (0.5% TritonX-100, 20 mM Tris pH 7.4, 10% Sucrose) NEP activity assay was performed according to Miners et al. (2008b (link)) with minor modifications utilizing the anti-NEP antibody AF1182 (R&D Systems, Minneapolis, Minn., USA) and 5 μM MCA-RPPGFSAFK(DNP)-OH fluorogenic peptide substrate (R&D Systems, Minneapolis, Minn., USA). Fluorescence was measured with an excitation wavelength of 320 ± 10 nm and an emission wavelength of 405 ± 10 nm in a Safire2 Fluorometer (Tecan, Crailsheim, Germany) as described above. Unspecificity of the assay was 22.1% (± 2.4%, p ≤ 0.001) as measured in presence of 10 μM thiorphan (Santa Cruz Biotechnology, Dallas, USA) (Supplementary Figure 1A).

After cells were lysed in lysis buffer (0.5% TritonX-100, 20 mM Tris pH 7.4, 10% Sucrose) IDE activity assay was performed as described in Miners et al. (2008a (link)) with minor modifications utilizing the anti-IDE antibody ST1120 (Calbiochem, Darmstadt, Germany) and 10 μM MCA-RPPGFSAFK(DNP)-OH fluorogenic peptide substrate (R&D Systems, Minneapolis, Minn., USA). Resulting fluorescence was measured at an excitation wavelength of 320 ± 10 nm and an emission wavelength of 405 ± 10 nm in a Safire2 Fluorometer as described above.

Measurement of NEP activity was performed as published in Miners et al. [84 (link)], with minor modifications utilizing the anti-NEP antibody AF1182 (R&D Systems, Minneapolis, MN, USA) and 5 μM MCA-RPPGFSAFK(DNP)-OH fluorogenic peptide substrate (R&D Systems, Minneapolis, MN, USA). Cells were chemical lysed in lysis buffer containing 0.5% TritonX-100, 20 nM Tris pH 7.4, 10% sucrose and fluorescence at an excitation wavelength of 320 ± 10 nm, and an emission wavelength of 405 ± 10 nm was measured in a Safire2 Fluorometer (Tecan, Crailsheim, Germany).