Af 1079

All animal protocols were reviewed and approved by the intramural Ethics Committee on Humane Treatment of Laboratory Animals of Nanjing Medical University. The mice were maintained in an SPF environment with 12 h light/dark cycles and ad libitum access to food and water. To induce atherosclerosis, 8-wk male Apoe^−/− mice were fed a Western diet (D12109, Research Diets, New Brunswick, NJ, United States) for 8 weeks as previously described (Zhang et al., 2020 (link)). An anti-Neo1 antibody (5 μg per injection, R&D, AF 1079) or isotype IgG was injected intravenously every day from week 5 of Western diet feeding till the mice were sacrificed. The animals were euthanized by pentobarbital sodium (100–120 mg/kg) to obtain their samples. Atherosclerotic lesions were gauged by en face analysis of the whole aorta and by cross-sectional analysis of the proximal aorta essentially described previously (Liao et al., 2020 (link); Huangfu et al., 2021 (link)).

E11.5 heads were lysed in 50 mM Tris pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1× Pefabloc SC PLUS protease inhibitor (Roche). Primary antibodies used include goat anti-Boc (1:1000; R&D, AF2385), goat anti-Dcc (1:1000; Santa Cruz, SC-6535), rabbit anti-Flk1 (1:2000; Cell Signaling; as described in Gu et al., 2003 (link)), goat anti-neogenin 1 (1:1000; R&D, AF1079) and rat anti-Ntn1 (1:500; R&D, AF1109). Each blot was performed twice using independent lysates.