0) for 2 min to reduce

the pH of the vascular bed, (4) 6 % CCSN solution for 3 min to label the surface of VECs, (5) MES for 1 min to wash out unbound CCSN, (6) 1 % sodium polyacrylate in MES for 2 min to cross-link CCSN and VEC plasma membrane, and (7) 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer [25 mM HEPES, 250 mM sucrose, 1 mM ethylenediaminetetraacetic acid (EDTA), pH 8.0] for 3 min to flush the vasculature. After perfusion, the left kidney was removed and minced with a razor blade in a plastic dish at 4 °C and then placed in 5 ml HEPES buffer. Homogenization was carried out for 2 min at 14,000 rpm (Polytron PT1200; Kinematica, AG, Switzerland). Vactosertib cost The MDV3100 research buy homogenate was filtered through a 40-μm nylon monofilament net, and the filtrate was then fractionated by Nycodenz (Axis-Shield plc, Scotland) gradient centrifugation as follows: the filtered homogenate was diluted with an equal volume of 1.02 g/ml Nycodenz, and the total volume of 5 ml mixture was layered onto

a 55–70 % Nycodenz Protein Tyrosine Kinase inhibitor gradient by placing 2.0 ml of 70 %, 1.5 ml of 65 %, 1 ml of 60 %, and 1 ml of 55 % Nycodenz in a 12-ml centrifuge tube. The tube was topped off with HEPES buffer and centrifuged at 15,000 rpm for 30 min at 4 °C in a swinging bucket rotor (P40ST; Hitachi High Technology, Japan). After centrifugation, the supernatant was removed, and the CCSN-labeled membrane fraction was collected at the bottom as a pellet. The pellet was then resuspended in 1 ml MBS. Then, an equal volume of 1.02 g/ml Nycodenz was added to the solution, and a second centrifugation was performed at 30,000 rpm for 60 min at 4 °C (CP80β; Hitachi High Technology, Japan), using a 80–60 % Nycodenz gradient (1.5 ml of 80 % Cell press and 0.7 ml of 75, 70, 65, and 60 % Nycodenz). The CCSN-coated membrane was collected as a pellet and was washed in 1 ml MBS buffer in a microfuge tube at 14,000g for 30 min. The CCSN was resuspended in 100 μl of 2 % sodium dodecyl sulfate (SDS) in 50 mM Tris buffer (pH 7.4) and sonicated at 50 Hz for 30 s to detach the CCSN from the VEC membrane. The suspension

was heated at 100 °C for 5 min to solubilize proteins, and the silica was separated by centrifugation at 14,000g for 15 min. Histological examination After perfusion of the CCSN beads, parts of the kidneys were fixed in 10 % formalin and embedded in paraffin for light-microscopic examination. Small kidney blocks of approximately 1 mm3 were fixed in 2.5 % glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) overnight for electron microscopy. Sections of the kidneys were stained with periodic acid-methenamine (PAM) to demonstrate binding sites of the CCSN beads by light microscopy. The glutaraldehyde-fixed blocks were postfixed for 1 h in 1 % OsO4 in 0.1 M phosphate buffer and then embedded in epoxy resin.