Nrp 1 rabbit monoclonal antibody

The target protein expression levels were examined using Western Blotting as described previously. For TECs derived from the kidney of CLP rats, protease inhibitor containing RIPA buffer (Sigma) was used to extract the total protein. For samples derived from NRK52E in vitro, membrane proteins and cytoplasm proteins were firstly separated with Subcellular Protein Fractionation Kit for Cultured Cells (Thermo Scientific) in accordance with the manufacturer's instructions; then, the proteins from either membrane or cytoplasm were prepared with protease inhibitor containing RIPA buffer (Sigma). The protein lysates were separated with SDS-PAGE and transferred to the PVDF films (Millipore). The PVDF films then were cut into pieces according to the molecular weights of the target proteins and incubated with the primary antibodies (Ago2 rabbit monoclonal antibody, 1 : 1000 (Novus); Nrp-1 rabbit monoclonal antibody, 1 : 1000 (Novus); and His-tag mouse monoclonal antibody, 1 : 1000 (MBL)) at 4°C for 12 h. Then, either AlexaFluor 680/790-labeled goat anti-rabbit IgG antibody (1 : 10000, LI-COR Biosciences) or AlexaFluor 680/790-labeled goat anti-mouse IgG antibody (1 : 10000, LI-COR Biosciences) was used as the second antibody, and the blots were visualized by LI-COR Odyssey Infrared Imaging System (LI-COR Biosciences). Quantity One was used to quantify the blots.

The immunofluorescent assay was carried out in the present study as described before. In brief, the cell climbing films were fixed in 4% paraformaldehyde with 0.1% Triton X-100 for 30 min at 4°C. Relevant primary antibodies (Ago2 rabbit monoclonal antibody, 1 : 200 (Novus); Nrp-1 rabbit monoclonal antibody, 1 : 200 (Novus)) and fluorescently labeled probe (LysoTracker Red DND-99 (YeasenBio)) were used to incubate with the films overnight at 4°C according to the manufacturers' instructions. The films then were incubated with selected secondary antibodies (FITC-conjugated goat anti-rabbit IgG antibody and TRITC-conjugated goat anti-rabbit IgG antibody) specifically for 90 min in the dark. The nuclei were stained using 4′,6-diamidino-2-phenylindole. The images were captured using a laser confocal microscope (Olympus).