Goat anti rabbit ir dye 800

For Western blotting, cells were grown in the presence of plain synthetic medium with 2% raffinose or 2% galactose (supplemented as indicated with 1% DMSO [dimethyl sulfoxide] or 1% DMSO + 50 μM TSA) for 16 h to log phase and harvested by centrifugation. Cells were lysed with 0.1 M NaOH and resuspended in reducing sample buffer. The equivalent of 1–2 OD₆₀₀ of culture was separated on a 15% SDS–PAGE, blotted onto a nitrocellulose membrane, and labeled with an antibody against anti-acetylated proteins (Cell Signaling #9441) at 1:1000 dilution and anti-Fibrillarin antibody (ThermoFisher Scientific 38F3) at 1:2000 dilution. Secondary antibodies were goat anti–mouse-Alexa680 (ThermoFisher Scientific A-21057) used at 1:10,000 dilution and goat anti–rabbit-IRDye800 (Rockland) used at 1:5000 dilution. Staining was visualized on an Odyssey CLx Infrared Imaging System (Li-Cor).

To quantify the level of Mas receptor protein expression during development, retinal tissue was collected from P1, P15, P21, and P60 wild-type mice in cold RIPA buffer (Sigma, St Louis, MO) supplemented with protease inhibitors and were homogenized by sonication. Homogenized tissues were centrifuged at 12,000 ×g for 15 min at 4 °C, and the supernatant was collected. The protein concentration was detected with the Bio-Rad Protein Assay kit (Hercules, CA). Forty microns of protein samples were loaded and separated on 4–12% gradient gels for 1 h at 120 V in Tris-glycine buffer and electrophoretically transferred onto polyvinylidene difluoride membrane. Immunodetection was performed on blots blocked in fluorescence blocking buffer for 1 h (Rockland Immunochemicals, Gilbertsville, PA) and then incubated with primary (rabbit anti-Mas; 1:2,000; Alomone Laboratories), and secondary (goat anti-rabbit IR Dye 800; 1:5,000; Rockland Immunochemicals) antibodies. β-Actin immunodetection was used as a loading control. Immunoblots were visualized by using a Li-Cor odyssey infrared imager (Odyssey; Lincoln, NE). We used Image J software to quantify the relative level of the Mas protein after normalizing it to β-actin.