Anti prr antibody

High affinity protein A-agarose (Abcam) was incubated with rabbit antisera anti-E V-ATPase subunit. The antibody was crosslinked to protein A-agarose using DMP (Sigma) as we have described [24 (link)]. EVs from three-day conditioned media from one 100 cm tissue culture plate were homogenized in 200–500 μl solubilization buffer (SB; 20 mM Tris/HCl pH 7.4, 5 mM Sodium Azide, 1 mM EDTA, 1 mM dithiothreitol, 1.5% n-octyl β glucopyrannoside, 0.6% CHAPS, 10% glycerol plus PMSF, and a cocktail of addition protease inhibitors). EVs were then homogenized by 15 passes in a 1 ml, tight fitting, glass homogenizer. Samples were incubated with 15 μl of the antibody conjugated protein A beads for 2 hours at 4 degrees Celsius, then washed 3X in 0.5 ml ice cold NET-gel buffer (50 mMTris/HCl pH 7.4, 150 mM NaCl, 0.1% Nonidet P-40, 1 mM EDTA, 0.25% gelatin, 5 mM NaAzide). The beads were then resuspended in 30 μl SDS-PAGE sample buffer, heated to 95 degrees Celsius, and the soluble material was separated by SDS-PAGE, blotted to nitrocellulose and probed with anti-PRR antibody (AbCam).

Cardiac fibroblasts chamber slides were used to detect PRR and YAP protein expression change in different treatments. After all of experiments PRR protein expression and YAP expression were detected by immunofluorescence staining. Cardiac fibroblasts in each group were fixed with 4% paraformaldehyde, blocked with 10% BSA and stained with anti-PRR antibody(diluted ratio 1:250, Abcam, Cambridge, MA) and YAP (diluted ratio 1:200, Cell Signaling Technology, Danves MA, USA) overnight at 4 ℃. PRR protein combined with specific primary antibody was bonded with FITC-conjugated IgG and YAP protein combined with specific primary antibody was bonded with TRITC-conjugated IgG. Cell nucleus were stained with 6-diamidino-2-phenylindole (DEPI) and samples were sealed with anti-fluorescence quenching agent (Abcam, Cambridge, MA). Immunofluorescence intensity was visualized under a Leica fluorescence microscope.

A total of 800 μg of whole cell homogenates from NRK-52E cells stimulated by prorenin (100 pmol/L, 48 h) was used for co-immunoprecipitation. Homogenates were precleared with protein A-agarose (Pierce, Rockford, IL) for 1 h at 4 °C. The supernatants were saved and incubated with a rabbit polyclonal anti-(P) RR antibody (4 μg, Abcam, Cambridge, UK) or rabbit IgG (2 μg, Sigma, MO) as a negative control at 4 °C overnight on a rotator. Protein A-agarose (50 μL, Pierce, Rockford, IL) was added to the complex and incubated for 4 h at 4 °C. The agarose pellets were washed three times with 1 mL of ice-cold lysis buffer (Cell Signaling Technology, Boston, MA) with a protease inhibitor cocktail (Cell Signaling Technology, Boston, MA) and one time in 1 mL of ice-cold PBS buffer. Bound proteins were extracted with 30 μL of 2× laemmli sample buffer (Bio-Rad) supplemented with 5% β-mercaptoethanol. The samples were heated at 95 °C for 5 min, and the supernatants were saved for Western blotting.