Ab12193

RSV, Suramin, U0126 and the SIRT1 Assay Kit (Sigma-Aldrich, USA); methyllycaconitine (MLA) (Tocris Bioscience, UK); TRIzol reagent (Invitrogen, China), SYBR Green PCR master mix (Applied Biosystems, USA); ECL Plus reagent (Merck Millipore, Germany); the near-infrared amyloid-β fluorescent CRANAD-58 probe (ab146926, Abcam Inc., USA); rabbit monoclonal anti-p-Erk1/2 and rabbit monoclonal anti-Erk1/2 antibodies (4370 and 4695, Cell Signaling Technology (CST) Inc., USA); rabbit monoclonal anti-SIRT1 antibody and anti-SYP (synaptophysin) (ab12193 and ab8049; Abcam Inc., USA); rabbit polyclonal anti-α7 nAChR and mouse monoclonal anti-β-actin antibodies (sc-58607 and sc-376421, Santa Cruz Inc., USA); rabbit polyclonal anti SNAP-25 (synaptosomal-associated protein 25), rabbit polyclonal anti CREB, rabbit polyclonal anti CREB (phosphpo-Ser133), rabbit polyclonal anti GFAP and rabbit polyclonal anti-Iba-1 (ionized calcium binding adaptor molecule 1) antibodies (GTX113839, GTX112846, GTX130379, GTX108711 and GTX100042, Gentex Inc., USA) and horseradish peroxidase-conjugated secondary antibody (7076s and 7074s, CST Inc., USA) were obtained from the sources indicated.

The brain sections and coverslips were fixed with 4% paraformaldehyde. Then, they were incubated in 0.1% Triton X-100 for 5 min. The slides were then incubated with primary antibodies including Iba-1 (SC-98468, 1 : 50, Santa Cruz), myeloperoxidase (MPO, SC-16128, 1 : 50, Santa Cruz), 8-hydroxydeoxyguanosine (8-OHdG, ab62623, 1 : 100, Abcam), SIRT1 (Ab12193, 1 : 100, Abcam), NeuN (MAB377, 1 : 200, EMD Millipore). After several times washes, they were incubated with appropriate secondary antibodies. The images were visualized by using an epifluorescence microscope.

Total cellular and nuclear protein was extracted from retinas and quantified using bicinchoninic acid assay kit. Homogenate in 2 × sodium dodecyl sulfate (SDS) sample buffer was boiled for 5 min, and then equal amounts of protein (40 μg) from each sample were subjected to electrophoresis on a 10% (v/v) SDS-polyacrylamide gel. After proteins were electroblotted to a polyvinylidene difluoride membrane, the membrane was blocked with Phosphate-buffered saline containing 5% dried non-fat milk or 3% BSA at room temperature for 1 h, and incubated with indicated primary antibodies (anti-ATF4, 1:1000, SC-200, Santa Cruz Biotechnology, Dallas, TX; anti-ATF6, 1:1500, ab37149; anti-GRP78/BiP, 1:2000, ab21685; anti-pIRE1α, 1:1000, ab48187, Abcam, Cambrigde, MA; anti-Nrf2, 1:1000, SC-722; Santa Cruz Biotechnology, Dallas, TX; anti-p38, 1:500, ab27986; anti-p-p38, 1:1000, ab4822; anti-JNK, 1:1000, ab59227; anti-p-JNK, 1:2000, ab124956; anti-p65, 1:600, ab7970; anti-p-p65, 1:2000, ab86299; anti-SIRT1, 1:2000, ab12193, Abcam, Cambrigde, MA) at 4 °C overnight, followed by incubating with the goat-anti-rabbit horseradish peroxidase-conjugated secondary antibody for 2 h. After incubation, membrane was washed three times, and the antigen-antibody complexes were visualized by the enhanced chemiluminescence system (PerkinElmer, Akron, OH).

Western blot was performed according to the manufacturer's specifications. The primary antibodies used were MVH (ab27591, Abcam, USA), OCT4 (ab18976, Abcam, USA), TNF-α (EPR22598-212, Proteintech, USA), IL-2 (26,156–1-AP, Proteintech, USA), IL-10 (Abcam, ab189392), TGF-β(ab179695, Abcam, USA). SIRT1(ab12193, Abcam, USA), SIRT3 (ab189860, Abcam, USA), p21(28,248–1-AP, Proteintech, USA), p53 (10,442–1-AP, Proteintech, USA), GAPDH (ab181602, Abcam, USA). All the HRP secondary antibodies were obtained from Affinity Biosciences. After accordingly antibody incubation and washing with TBST, PVDF membranes were imaged using an EasySee Western blot kit (DW101–01, TransGen, China). Images AI600 and Image J were used to scan and analyze the images.

ChIP analysis was conducted with a ChIP Assay Kit (Millipore) following the manufacturer’s instructions. Sorted monocytes were cross-linked in 1% formaldehyde for 10 min at room temperature, and then, we added glycine to stop the reaction. The fixed cells were washed with 20 ml ice-cold phosphate-buffered saline twice and then lysed. The cell lysates were centrifuged and resuspended before being sonicated to generate 500- to 1,000-bp fragments. After that, the indicated antibodies were added and incubated with the sheared DNA. The immune compounds were precipitated with protein A agarose beads, and then washed and eluted. The precipitated DNA was purified, and then, the target DNA was amplified by qPCR. The primers used are presented in Supplementary Table S3. Anti-PPAR-γ (Abcam, ab41928) and anti-SIRT1 antibodies (Abcam, ab12193) were of ChIP grade and were purchased from Abcam. An anti-histone H3ac (pan-acetyl) antibody (pAb) (cat. no. 39139) and an anti-histone H4ac (pan-acetyl) antibody (pAb) (cat. no. 39243) were purchased from Active Motif.

Protein expression and location was examined by in situ immunofluorescence staining. Sections were incubated in PBS containing 5% normal goat serum, 1% bovine serum albumin (BSA) and 0.5% Triton X-100 for 1 h to block non-specific binding, followed by incubation with primary antibodies (anti-GRP78/BiP, 1:400, ab21685; anti-p-JNK, 1:100, ab124956; anti-SIRT1, 1:50, ab12193, Abcam, Cambrigde, MA) overnight at 4 °C. After three washes with PBS, sections were incubated with goat anti-rabbit IgG H&L Alexa Fluo^® 555 (1:600, ab150086, Abcam, Cambrigde, MA) for 2 h at room temperature. After washing with PBS, sections were mounted with VECTASHIELD mounting medium with DAPI (VECTOR LABORATORIES, Burlingame, CA) and examined under a fluorescent microscope.