Sc 17845

In IP, the lysate protein (500 μg) was mixed with 2 μg protein G agarose antibody (16e266, Millipore, Billerica, MA, USA), and incubated and rotated overnight at 4° C. Proteins were collected as IP production after washing buffer three times. The succinylation signal was detected and separated on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred onto a polyvinylidene difluoride (PVDF) membranes (Millipore, USA), and incubated with primary antibodies: NOX-1 (1:100, ab121009, Abcam), EBP50 (1:100, 3394, Abcam or 1:50, sc-271552, Santa Cruz Biotechnology), p47phox (1:100, ab166930, Abcam or 1:50, sc-17845, Santa Cruz Biotechnology) overnight at 4° C. Membranes were washed with TBST and incubated with HRP-conjugated secondary antibody (sc-2004, sc-2005, 1:2000, Santa Cruz Biotechnology) at room temperature for 1 h. Membrane was developed with ECL (Promega, USA).

Cell were washed with PBS and fixed with 4% paraformaldehyde supplemented with 0.25% Tris-X100 at room temperature for 10 min. After blocking with PBS supplemented with 5% BSA for 2 h at room temperature, cells were incubated with NOX-1 (1:200, ab55831, Abcam), EBP50 (1:1000, 3394, Abcam or 1:200, sc-271552, Santa Cruz Biotechnology), p47phox (1:200, ab166930, Abcam or 1:200, sc-17845, Santa Cruz Biotechnology) at 4° C overnight. Cells were incubated with secondary peroxidase conjugated goat anti-rabbit IgG (1:100, Santa Cruz Biotechnology) antibody for 2 h at room temperature, after washing with PBST for 15 min. Cells were stained with DAPI for 15 min at darkness, after washing with PBST for 15 min. Cell samples were observed using fluorescence microscope (Zeiss Axio Observer A1, Germany).

Western blotting was used to detect protein expression in the cerebrovascular endothelium as performed in the past study [29 (link)]. Isolated brain vessels were homogenized and processed, and protein concentrations were measured with a bicinchoninic acid protein assay kit (Pierce Biotechnology, Inc.). Primary antibodies to tumor necrosis factor-α (TNF-α) (1 : 1000, 11948, Cell Signaling Technology), interleukin-1β (IL-1β) (1 : 5000, ab9722, Abcam), interleukin-6 (IL-6) (1 : 200, ab7737, Abcam), vascular cell adhesion molecule-1 (VCAM-1) (1 : 2000, ab134047, Abcam), macrophage chemoattractant protein-1 (MCP-1) (1 : 1000, ab25124, Abcam), gp91^phox (1 : 1000, ab129068, Abcam), p47^phox (1 : 1000, sc-17845, Santa Cruz, Inc.), p67^phox (1 : 1000, sc-374510, Santa Cruz, Inc.), p22^phox (1 : 1000, sc-271968, Santa Cruz, Inc.), and β-actin (1 : 5000, A5060, Sigma-Aldrich) were incubated on the membrane at 4°C overnight. The membranes were washed three times with PBS for 10 minutes each and reincubated with secondary antibody (1 : 10,000, Invitrogen) for 1 hour at room temperature. Protein expressions were analyzed with an enhanced chemiluminescence kit (Millipore). Quantification of relative target protein expression was performed by ImageJ 1.42 (National Institutes of Health).