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Collagen hydrogels, neprilysin standards (20-50-100 ng/lane) or medium with released neprilysin (20 μl per lane) were denatured (10 min 70°C with reducing agent) and loaded onto 10% Bis-Tris polyacrylamide gel (Invitrogen) and electrophoresis was performed for 35 min at 200 V. Samples were electrotransferred onto PVDF membranes for 20 min at 25 V in a semi-dry transfer cell (Thermo Fisher Scientific). Blotting was done by using WesternBreeze Chemiluminescent immunodetection system (Invitrogen). Blots were blocked with blocking buffer for 30 min and incubated overnight on a shaker at 4°C with primary antibodies against neprilysin (abcam ab210932; 1:1000). Subsequently, blots were briefly washed and incubated with alkaline phosphatase-conjugated secondary mouse (neprilysin) for 30 min at room temperature. Afterward, the blots were briefly washed again, incubated for 15 min in CDP-Star chemiluminescent substrate solution (Roche) and visualized with a cooled CCD camera (SearchLight, Thermo Fisher Scientific).

Western blot analysis was performed as previously described by us (21 (link)). Platelet samples (−80°C) were thawed and tubes dissolved in 100 μL ice-cold PBS containing a protease inhibitor cocktail (P-8340; Sigma). Samples were then sonicated using an ultrasonic device, centrifuged at 14,000 × g for 10 min at 4°C; the extracts were denatured (10 min, 70°C), and 18 μg was loaded onto 10% bis–tris SDS-polyacrylamide gels (Thermo Fisher Scientific, Vienna, Austria), separated for 35 min at 200 V and finally electrotransferred to nylon-PVDF Immobilon-PSQ membranes for 20 min at 30 V in 20% methanol blotting buffer. Next, blots were blocked for 30 min in blocking buffer; incubated with primary antibody against APP (Abcam ab32136, 1:2,000, Cambridge, UK), or CD41 (Abcam ab63323, 1:2,000), or actin (1:1,000, A2066; Sigma, Vienna, Austria) at 4°C overnight; washed; and then incubated in alkaline phosphatase–conjugated anti–rabbit IgG for 30 min. After washing, bound antibodies were detected using an enhanced chemiluminescence system and visualized by using a cooled CCD camera (SearchLight; Thermo Fisher Scientific).

Western blots were performed as described by us previously [14 (link)]. Briefly, the tissue was dissolved in 200 µL ice-cold PBS with a protease inhibitor cocktail (P8340, Sigma, Hamburg, Germany), homogenized by using an ultrasonic device (Branson sonifier 250, Danburry) and centrifuged at 16,000× g at 4 °C for 10 min. Protein was determined by the Bradford assay. The non-denatured supernatants (25 µg) were loaded onto a 10% Bis-Tris polyacrylamide gel (Invitrogen, Waltham, MA, USA) and were electrophoresed for 25 min at 200 V. Samples were electrotransferred to nylon PVDF (Immobilon-PSQ membranes (Millipore, Burlington, MA, USA) for 90 min at 30 V with 20% methanol blotting buffer (Invitrogen). Blots were blocked for 30 min with blocking buffer, then incubated overnight at 4 °C with the primary serpinin antibody (1:1000; gift from Peng Loh, National Institutes of Health, Bethesda, MD, USA; polyclonal antibody, raised in rabbits) or overnight at 4 °C with a rabbit anti-actin antibody (1:1000, Sigma-Aldrich, St. Louis, MO, USA). Blots were washed and incubated with alkaline phosphatase-conjugated anti-rabbit antibodies for 30 min at room temperature. After washing, blots were incubated (free-floating) in CDP-Star chemiluminescent substrate solution (Invitrogen) for 10 min and the signal was visualized (exposure time 1200 s) with a cooled CCD camera (SearchLight, Thermoscience).