Anti goat irdye 800cw

Fixed hMPCs were permeabilized with 0.1% Triton in PBS. Cells were incubated in primary antibodies, specific for PYY (ab22663, Abcam, Supplementary Figure S2) and Y1r, Y2r, and Y5r diluted (PYY, 1:1000; Y receptors, 1:100) in Odyssey Blocking Buffer (LI-COR), overnight at 4°C. Cells were washed with 0.1% PBST and incubated at room temperature in secondary antibody (IRDye^® 800CW anti-rabbit, LI-COR or IRDye^® 800CW anti-goat, LI-COR) diluted 1:400 in Odyssey Blocking Buffer. Fluorescence intensity, representing protein levels, was measured using the Odyssey Imaging System (LI-COR). Protein levels were normalized to number of cells via Hoechst 33342 staining and counting using the Celigo^® Imaging Cytometer (Nexcelom Bioscience).

Diethylamine NONOate (NO donor) was purchased from Enzo Life Sciences (Cat # ALX-430-014-M005, Farmingdale, NY, United States). L-NAME (NOS inhibitor, Cat # N5751), Calphostin C (protein kinase C – PKC – inhibitor, Cat # 20785), and ODQ (soluble guanylyl cyclase inhibitor, 1H-(1,2,4)oxadiazolo[4,3-a]quinoxalin-1-one, Cat # O3636) were purchased from Sigma-Aldrich (Saint Louis, MO). A selective protein kinase G (PKG) inhibitor, KT5823, was purchased from TOCRIS (Cat # 1289, Bristol, United Kingdom).
We used a mouse anti-renin polyclonal IgG B-12 antibody (sc-81178) for detection of renin, and ß-actin was detected using a goat anti- ß-actin monoclonal IgG antibody (sc-1615), both purchased from Santa Cruz Biotechnology, (Santa Cruz, CA, United States). For western blot procedures the secondary antibodies used were the IR Dye 800CW anti-goat and mouse according to the primary antibody chosen (Li-Cor Bioscience, NE, United States). For immunofluorescence, we utilized a mouse anti-renin polyclonal IgG H-105 antibody (sc-22752–1:200), and secondary Alexa Fluor antibodies (Alexa fluor-488), both purchased from Life Technologies (Carlsbad, CA, United States). The mouse kidney cortical CD cells (M-1 cell line) were purchased from American Type Culture Collection (Cat: CRL-2038, ATCC, Manassas, VA, United States).

Western blotting was performed as described before.^{22 (link)} Briefly, protein extracts of mouse corneas were prepared in a radioimmunoprecipitation (RIPA) buffer supplemented with 1% SDS and Halt protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA). Four corneas were pooled and considered one biological replica. Protein concentration was determined by Bradford-based protein assay (Bio-Rad protein assay). Equal amounts of lysates (30 μg protein) were subjected to electrophoresis in 4% to 15% gradient SDS-PAGE gels (Bio-Rad, Hercules, CA, USA). Protein blots of the gels were blocked with Odyssey blocking buffer (OBB; Li-Cor Biosciences, Lincoln, NE, USA) and incubated overnight with primary antibodies: rabbit anti-α-SMA (ab5694, 1:2000 dilution in OBB; Abcam, Cambridge, MA, USA), goat anti-connective tissue growth factor (CTGF) (clone L-20, 1:500 dilution in OBB; Santa Cruz Biotechnology, Dallas, TX, USA), and mouse anti-β-actin (clone AC-15, 1:10,000 dilution in OBB; Santa Cruz Biotechnology). The secondary antibody used was anti-rabbit IgG IRDye 800CW, anti-goat IRDye 800CW, and anti-mouse IgG IRDye 680LT (1:10,0000 dilution in OBB; Li-Cor Biosciences). Blots were then scanned with the Odyssey Infrared Imaging System, and relative band intensity was quantified by Image Studio v2.0 software (Li-Cor Biosciences).