The left ventricular membranes were prepared as described previously [ 3, 25], and 100 μg of protein was incubated in duplicate with an ACE2-specific QFS, 7-methoxycoumarin-4-yl -acetyl-Ala-Pro-Lyss (2,4-dintirophenyl) (Auspep), 10 μM Z-Pro-prolinal (Auspep), with or without 100 μM EDTA in a total volume of 200 μl [ 3].
Human KL-6 levels were determined in duplicate with a PICOLUMI KL-6 kit (EIDIA, Tokyo, Japan), an electrochemiluminescence immunoassay (ECLIA) specific for human KL-6.
Strains were pinned in duplicate with a density of approximately 864 per plate.
Glucose was measured in duplicate with a YSI 2300 autoanalyzer (Yellow Springs Instruments; Yellow Springs, OH, USA).
These experiments were conducted in duplicate with a different batch of nanoparticles used for each experiment.
All samples were run in duplicate with a coefficient of variation between duplicates of <1.0 cycle.
Standing height was determined in duplicate with a standard stadiometer.
Samples were first screened in duplicate with a 1 10 dilution.
Concentrations of immunoglobulins IgG, IgA and IgM were determined in duplicate with porcine specific enzyme-linked immunosorbent assays (ELISA) according to the manufacturer's instructions (Bethyl, Laboratories Inc., Montgomery, TX USA).
Real-time PCR analysis of gene expression was performed in duplicate with specific internal oligonucleotide primers and the TaqMan probe (Universal ProbeLibrary-UPL, Roche, Mannheim, Germany).
