Ang 2

Western blot analyses were conducted as previously described [4 (link)] using anti-AT1R (Santa Cruz, 1 : 1000), -AngII (Novus, 1 : 1000), -Nox2 (BD transduction, 1 : 5000), -Nox4 (Santa Cruz, 1 : 5000), -nitrotyrosine (Cayman, 1 : 1000), -Ly6G (ebioscience, 1 : 1000), and -GAPDH (Santa Cruz, 1 : 5000) antibodies. Densities of the blots were quantified using the ImageJ program (NIH, Bethesda, MD).

Colon sections (5μm) were deparaffinized, rehydrated through a series of washes in graded ethanol and water, followed by an antigen retrieval step (by boiling the sections in 10 mM sodium citrate buffer, pH 6.0 for 20 min). Sections were then incubated in blocking solution (5% bovine serum albumin (BSA) + 0.3% Triton X-100 in PBS) for 1 h, followed by incubation overnight at 4°C with primary antibodies [p-ERK1/2, p-Akt (1:50 dilution), p-p38 MAPK and MAS-1R (1:100 dilution); Cell Signaling, USA, and Ang II (1/50 dilution); Novus Biological] diluted in 1% blocking solution. On the following day, sections were washed and incubated with secondary antibody conjugated to Alexa Fluor 555 (Goat anti rabbit SFX kit; Life Technologies,USA, 1:400 dilution) for 2 h at room temperature) in the dark. After washes in PBS sections were stained with 4’,6 diamidino-2- phenylindole and mounted. Images were captured on a ZIESS LSM 700 confocal microscope and fluorescence intensity estimated in defined fields using Image J software package.

Primary antibodies were against p19 (NB200‐106, Novus Biologicals, CO, USA), p21 (#2947, Cell Signaling Technology, Beverly, MA, USA), p53 (#2424, Cell Signaling Technology, USA), p16 (ab211542, Abcam, Cambridge, MA, USA), renin (#5250, Cell Signaling Technology, USA), Ang II (NBP1‐31127, Novus Biologicals, USA), ANP (#AB5490, Millipore, MA, USA; sc‐515701, Santa Cruz Biotechnology Inc., Dallas, TX, USA, USA), BNP (#DF6902, Affinity Biosciences, OH, USA; ab19645, Abcam, USA), GATA4 (#19530, Proteintech, IL, USA; sc‐25310, Santa Cruz Biotechnology Inc., USA), LC3B (#NB600‐1384, Novus Biologicals, USA), p62 (#39749, Cell Signaling Technology, USA), Bmi‐1 (#5856, Cell Signaling Technology, USA; 66161‐1‐Ig, Proteintech, USA), RING1B (#5694, Cell Signaling Technology), NF‐κB‐p65 (sc‐8008, Santa Cruz Biotechnology Inc., USA; #8242, Cell Signaling Technology, USA), p‐p65 (Ser536) (ab76302, Abcam, USA), IκB‐α (AF1282, Beyotime Biotechnology, Shanghai, China), p‐IκB‐α (Ser32) (sc‐8404, Santa Cruz Biotechnology Inc., USA), p‐Chk2 (Thr68) (PA5‐104715, Invitrogen Inc. CA, USA), SOD‐2 (NB100‐1992, Novus Biologicals, USA), HSC70 (10654‐1‐AP, Proteintech, USA), p‐ULK1 (Ser757) (#14202, Cell Signaling Technology, USA) and ULK1 (sc‐390904, Santa Cruz Biotechnology, USA). β‐actin (AP0060, Bioworld Technology Inc., MN, USA) was loading control for total protein.