Anti notch3

Cells were transfected with 20 nM siRNAs anti-Notch3 (Santa Cruz Biotechnology; Cat#sc-37135) or Pin1 (Santa Cruz Biotechnology; Cat#sc-36230) and the corresponding control scrambled siRNAs (Santa Cruz Biotechnology; Cat#sc-37007) using Neon transfection System (Life Technologies; Invitrogen) following the manufacturer's recommendations. Cells were analyzed 48 or 72 h after transfection.

Tissue samples were fixed and paraffinized as described^{25 (link)}. The 4 μm thick sections were prepared from paraffin-embedded tissues and immunostained with anti-CD45 (X16/99, Novocastra, Leica biosystems, Newcastle, UK, Cat# NCL-L-LCA), anti-Notch3 (Santa Cruz Biotechnology, Dallas, TX, USA, Cat#5539) or anti-CHOP (Cell Signalling, Danvers, MA, USA, Cat#2895) antibodies. After washes, secondary biotinylated antibodies were applied. Binding of antibodies was detected with the Mouse to Mouse HRP (DAB) Staining System (Scytek Laboratories, Inc., Logan, UT, USA) according to the manufacturer’s protocol. The analysis was conducted blindly.

Immunoblot analysis was carried out as previously described with slight modifications^{12 (link)}. Briefly, cell lysates were prepared in immunoprecipitation (IP) buffer (50 mM Tris-HCl [pH 7.5], 200 mM NaCl, 1% NP-40, 1% sodium deoxycholate with 1 mM PMSF, 1 μg/μl aprotinin, and 1 μg/μl leupeptin as protease inhibitors) and incubated on ice for 30 min. Total cell lysates (15 μg) were subjected to SDS-PAGE, followed by immunoblot detection with anti-Tuj1 (1:1,000, Millipore), anti-Ub (1:1,000, Millipore), anti-Notch3 (1:200, Santa Cruz Biotechnology), anti-CC3 (1:1,000, Millipore) or anti-β-Actin antibody (1:2,000, Santa Cruz Biotechnology). Based on the types of primary antibodies, the appropriate HRP-conjugated goat anti-mouse or anti-rabbit IgG (1:10,000, Enzo Life Sciences) or HRP-conjugated donkey anti-goat IgG (1:5,000, Santa Cruz Biotechnology) was used.

Following sacrifice, eyes were enucleated and fixed in 4% paraformaldehyde for 2 hours at 4°C. Retinas were dissected from the eye and incubated in blocking solution (0.5% Triton–X-100, 1% BSA in PBS) for 3 hours at room temperature. Retinas were stained with the following primary antibodies at 4°C overnight in PBLEC (1% Triton-X-100, 1 mM MgCl2, 1mM MnCl2, and 1mM CaCl2 in PBS [pH 6.8]); Biotinylated isolectin B₄ (1:50; Vector), anti-CD31 (1:100, BD Pharmingen), anti-NG2 (1:500, Millipore), anti-desmin (1:25, R&D), anti-αSMA-Cy3 (1:750, Sigma), anti-Notch3 (1:200, Santa Cruz), anti-collagen type IV (1:500, Cosmo. Bio), anti-laminin (1:500, Abcam), and 488-conjugated anti-GFP (1:200, Invitrogen). After washing with PBLEC, retinas were incubated with Alexa Fluor conjugated secondary antibodies (1:500, Invitrogen) in PBLEC. Stained retinas were flat mounted in 90% glycerol and confocal stacked images were acquired using a Nikon A1R microscope. Quantification of vessel density and pericyte coverage was performed using Image J on four 10× confocal stacked images per retina. Tip cells were defined as filopodia bursts and counted manually on four 40× images per retina.

Western blotting was performed as previously described (Zhao et al., 2014 (link)). The blots were probed with the following primary antibodies: anti-Notch2 (Cell Signaling Technologies) at 1:1000 dilution, anti-Notch3 (Santa Cruz Biotechnology) at 1:200, anti-NF-κB p65 (Cell Signaling Technologies) at 1:1000, anti-cyclin D1 (Cell Signaling Technologies) at 1:1000, anti-c-Myc (Abcam,) at 1:5000, anti- MMP-2 (Abcam) at 1:1000, anti-GAPDH (Affinity Biosciences, Cincinnati, OH, USA) at 1:5000, and anti-β-actin (KangChen, Shanghai, China) at 1:5000. Blots were detected using the Western Chemiluminescent HRP Substrate (Merck Millipore, Billerica, MA, USA) and visualized by FluorChem^®HD2 (ProteinSimple, San Jose, CA, USA).

Anti-JAG1 (Santa Cruz, sc-6011), anti-HTRA1 (R&D, MAB2916), anti-GAPDH (abcam, ab8245), anti-α-SMA (Sigma, C6198), anti-SM22α (abcam, ab137453), anti-NOTCH3 (Santa Cruz, sc-5593), anti-myosin (eBioscience, 14-6400), pSMAD2/3 (Cell Signaling, #8828) were used as primary antibodies. F-actin was stained with Alexa Fluor 568-conjugated phalloidin (Invitrogen).