Icam 1

Whole-cell lysates were prepared using RIPA buffer, size-fractionated through 4–12% gradient polyacrylamide gels (Thermo Fisher Scientific), and transferred to nitrocellulose membrane using Trans-Blot Turbo RTA Transfer Kit, Nitrocellulose (Bio-Rad). Membranes were incubated for 16 h with primary antibodies recognizing NFKB2 (Cell Signaling Technologies), CXCL10 (Abcam), MIF (Abcam), CR1 (Abcam), ICAM1 (Abcam), and ACTB (Santa Cruz Biotechnology), as described in Table 3. After incubation with the appropriate secondary antibodies, conjugated with horseradish peroxidase (HRP), the signals were detected by enhanced chemiluminescent reaction using SuperSignal West Femto (Thermo Fisher Scientific). Images were acquired with the Bio-Rad Universal Hood II Gel Doc System.

The abdominal aorta was isolated, fixed in 4% PFA for 12 min, opened in the longitudinal direction, and stained for ICAM1 (Abcam), VCAM1 (Abcam), or VE-cadherin (Abcam) in combination with DAPI. En face sections were obtained on a Leica SP8 confocal microscope, and all the analyses were performed with Image J [15 (link)].

Protein extracts from VSMCs were quantified using a commercial reagent Bradford 1 × Dye Reagent (Bio-Rad) and normalized to 0.5 μg/μl for all samples. Protein extracts were separated by SDS-PAGE and transferred to PVDF membrane. After blocking with 5% milk in TBST, the membranes were incubated with primary antibodies against VCAM-1 (1:1000, Epitomics), ICAM-1 (1:1000, Abcam), MCP-1(1:500, Santa Cruz), SIRT1 (1:1000, Merck Millipore), NF-κB p65 (1:1000, GeneTex), Acetyl-NF-κB p65 (Lys310) (1:200, Cell Signaling Technology), GAPDH (1:1000, Cell Signaling Technology) or Lamin A/C (1:1000, Cell Signaling Technology) at 4°C overnight. After incubating with HRP-conjugated secondary antibody (1: 20 000, Abcam), the blots were visualized using GE ImageQuant™ LAS 4000 detection system. Band intensities were quantified with Image Pro Plus 6.0 software.
Lysates were precleared with Protein A/G PLUS-Agarose (Santa Cruz) to reduce nonspecific binding. The supernatants were immunoprecipitated with indicated antibodies at 4°C overnight, followed by incubation with Protein A/G PLUS-Agarose beads for 2 h. The agarose beads were then collected by centrifugation, washed with the lysis buffer, and resuspended in sample buffer. Bound proteins were resolved by SDS-PAGE followed by Western blot analysis as described above. These experiments were replicated three times.

Brain samples were homogenized and sonicated in lysis buffer. Total protein was measured by a NanoDrop apparatus (MaestroGen, Las Vegas, NV, USA). 20 μg protein were size-fractionated using any-kD Mini-Protean TGX gel electrophoresis and then transferred to a nitrocellulose membrane, and membranes were blocked and washed in TBS-T, and incubated overnight with AC, ACC1, ACC2, PC, PCC, MCC, presynaptic synapsin I, postsynaptic PSD95, PSD93, IL-17, IL-6, TNF-α, NFkB, GFAP, GAP43, ICAM-1, BDNF, CXCL 16, OPG and MMP-9 antibodies (Abcam, Cambridge, UK). The following day, membranes were washed and then incubated with horseradish peroxidase-conjugated goat anti-rabbit (Abcam, Cambridge, UK) or goat anti-mouse (Abcam, Cambridge, UK) secondary antibody (diluted 1:1000) for 1 h at room temperature. Protein loading was controlled with an anti-β-actin antibody (Abcam, Cambridge, UK). Protein levels were analyzed densitometrically using an image analysis system (Image J; National Institute of Health, Bethesda, MD, USA), corrected with values determined on β-actin blots, and expressed as relative values compared with the control group.

Soluble E-selectin, L-selectin, ICAM-1, and VCAM-1 in macaque sera were assessed using E-selectin (Abcam, Cambridge, UK), VCAM-1 (Uscn, Wuhan, China), and ICAM-1 (Abnova, Taoyuan, Taiwan) ELISA kits following the manufacturer instructions. Serum samples were diluted 1:10. All samples and standard dilutions were assayed in duplicate, the optical densities measured using a microplate spectrophotometer (Multiskan Go; Thermo scientific, Waltham, MA), and the concentrations of cell adhesion molecules were determined from a standard curve.

The brain was sectioned serially in 300 μm increments from the bregma to lambda, both sides of the PVN tissues were isolated by the use of a punch-out technique with a cryostat [33 (link), 34 (link)], and the PVN tissue was stored at − 80 °C until use. Western blotting analysis was performed in the same manner as previously described [6 (link)]. The protein levels were determined from tissue homogenate obtained from the PVN for the following antibodies: NLRP3 (1:2000, Santa Cruz, CA, USA), ASC (1:500, Santa Cruz, CA, USA), pro-caspase-1 (1:2000, Abcam, MA, USA), IL-1β (1:500, Santa Cruz, CA, USA), CXCR3 (1:2000, Abcam, MA, USA), VCAM-1, ICAM-1 (1:2000, Abcam, MA, USA), and CCL2 (1:2000, Santa Cruz, CA, USA), Iba-1 (1:500, Santa Cruz, CA, USA). The β-actin antibody was used as an internal standard, and band densities were analyzed with NIH ImageJ software.