Anti netrin 1

To study the localization and effect of MUC4 knockdown on netrin-1 expression, immunofluorescence double staining assay was performed. Cells were grown on sterile coverslips in 12-well plates, fixed in 3.7% paraformaldehyde for 20 min at room temperature, and incubated with anti-MUC4 1G8 and anti-Netrin-1 (Santa Cruz Biotechnology; 1:50 dilution). The secondary antibodies used were Alexa Fluor 488 and Alexa Fluor 594 (Invitrogen; 1:1000 dilution). 4′-6-Diamidino-2-phenylindole (DAPI) was applied to the samples after the final wash to visualize the nuclei. Images were visualized using a Zeiss fluorescence microscope.

Protein was extracted from aorta or BAECs by lysis using 1x RIPA buffer (Upstate, Temecula) containing protease inhibitors and phosphatase inhibitors. Lysate was centrifuged at 14,000 g for 10 min at 4°C and supernatant was used for protein estimation. Equal amount of protein (20 μg) was separated by electrophoresis on a 10% SDS-polyacrylamide pre-cast gel and transferred to polyvinylidene difluoride membrane. Blots were blocked using 2% bovine serum albumin (Sigma), incubated with their respective primary antibodies (anti-arginase 1, 1:10000; anti-arginase 2, 1:250; DCC, 1:500; anti-netrin-1, 1:500, Santa Cruz Biotechnology, Inc; anti-COX2, 1:1000, Abcam; anti- β-actin, 1:5000, Sigma-Aldrich; anti-p44/42 MAPK ERK1/2 and anti-phospho p44/42/MAPK ERK1/2 at Thr²⁰² and Tyr²⁰⁴, 1:1000; anti-eNOS and anti-phospho-eNOS at Ser¹¹⁷⁷, 1:1000; cleaved caspase-3, 1:1000; anti-NFkβ p65, 1:1000, Cell Signaling Technology, Inc) overnight at 4°C. After incubation with secondary antibodies, signals were visualized using an enhanced chemiluminescence kit (Amersham, Piscataway, NJ, USA). Bands were observed using Kodak image analyzer or Gene Snap (Syngene, Frederick, MD). Densitometric analysis was carried out using the Gene Snap software, results normalized to β-actin protein and expressed as arbitrary unit.