Anti annexin a1

Aβ42 peptide was purchased from American Peptide Company (Sunnyvale, CA, USA) and was prepared as previously described (Byun et al., 2015). It was dissolved in hexafluoroisopropanol for 72 h at room temperature (RT) and lyophilized. The peptide was then dissolved again in dimethylsulfoxide. Anti‐ZO‐1, anti‐Claudin 5 (Thermo Fisher Scientific, Waltham, MA, USA), anti‐GAPDH (Abcam, Cambridge, MA, USA), anti‐GFP (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), anti‐CD31 (R&D Systems, Minneapolis, MN, USA), antiphospho CREB (Cell signaling Technology, Danvers, MA, USA), anti‐CREB (Cell signaling Technology), and anti‐Annexin A1 (Invitrogen, Carsbad, CA, USA) were used for Western blot analysis, and Anti‐ZO‐1 (Thermo Fisher Scientific) was used for immunofluorescence images. Y27632, MK801, and l‐glutamate were purchased from Sigma‐Aldrich Co. (St. Louis, MO, USA), Rhosin was purchased from Merck Millipore (Billerica, MA, USA), and human Annexin A1 (ANXA1) recombinant protein was purchased from MyBioSource (San Diego, CA, USA). For experiments, bEnd.3 cells and/or pericytes were treated with Aβ42 (2 μm and/or 5 μm; from 0 to 24 h, differently for each experiment), ANXA1 (1 μg mL⁻¹ and/or 2 μg mL⁻¹; for 24.5 h), Y27632 (30 μm; for 24 h), Rhosin (10 μm; for 24 h), l‐glutamate (30 μm; for 30 min or 24 h), and MK801 (10 μm; for 30 min or 24 h).

Antibodies used in this study for immunoblot include: anti-phospho-ERK1/2 (#4370, Cell Signaling Technologies), anti-ERK1/2 (sc-514302, Santa Cruz), anti-Annexin A1 (#71-3400, Invitrogen), anti-β-actin (A5316, Sigma-Aldrich), and HRP-labeled secondary goat anti-mIgG (#31430, Invitrogen) and anti-rIgG (#31460, Invitrogen). Antibodies used in this study for flow cytometric staining and/or immunohistochemistry staining were: fluorescein-labeled anti-B220 (#103207, clone RA3-6B2, Biolegend), anti-CD19 (#115508, clone 6D5, Biolegend), anti-CD3e (#553061, clone 145-2C11, BD Biosciences), anti-CD11c (#117313, clone N418, Biolegend), anti-CD103 (#121413, clone 2E7, Biolegend), anti-IgD (#17-5993-82, clone 11–26, eBioscience), anti-CD3 (#100240, clone 17A2, Biolegend), anti-B220 (#103228, clone RA3-6B2, Biolegend) antibodies, and Hoechst 33,342 nucleic acid staining reagent (#H3570, Invitrogen) as well as HRP-labeled anti-NFκB (#MAB3026, Merck) antibody.
Genomic DNA extraction kit (#KN-T110005, KANEKA Co., Japan) and Taq DNA polymerase (#M0273, NEB Biolabs) were used for mice genotyping. All other biochemicals were purchased from Sigma-Aldrich.