Gtx628802

Immunoblot assays were conducted as described previously [28 (link)]. The following primary antibodies were used: anti-IGFBP3 (1:1000, GTX113364, GeneTex, Irvine, CA, USA), anti-cytochrome c (1:1000, 556,433, BD Biosciences, Franklin Lakes, NJ, USA), anti-cleaved caspase 3 (1:500, IMG-144A, IMGENEX, San Diego, CA, USA), anti-light chain 3B (1:500, LC3B, GTX127375, GeneTex), anti-NF-κB (1:1000, #6956, Cell Signaling), anti-phosphorylated NF-κB (1:1000, #3033, Cell Signaling), anti-IκBα (1:1000, ab76429, Abcam, Cambridge, UK), anti-phosphorylated IκBα S32 (1:1000, ab92700, Abcam), and anti-α-tubulin (1:5000, GTX628802, GeneTex). Protein levels were determined by measuring the intensity of bands on western blots using ImageJ (National Institutes of Health, Bethesda, MD, USA). Protein levels were normalized against α-tubulin as an internal control. The relative ratio was calculated by dividing the normalized protein levels in stably expressing cells with that in control cells.

Brain tissues were lysed in radioimmunoprecipitation assay buffer (Thermo Fisher Scientific). Proteins were then separated by SDS-PAGE and transferred to PVDF membranes (Millipore) using standard procedures [72 (link)]. The antibodies used were rabbit anti-HTR6 (1:500, Abcam #ab103016), rabbit anti-phospho-S6K (Thr389, 1:1,000, Cell Signaling Technology #9205), rabbit anti-S6K (1:1,000, Cell Signaling Technology #2708), rabbit anti-phospho-Akt (Ser473, 1:1,000, Cell Signaling Technology #9271), rabbit anti-Akt (1:1,000, Cell Signaling Technology #9272), rabbit anti-phospho-PKA (Thr197, 1:1,000, Cell Signaling Technology #4781), rabbit anti-PKA (1:1,000, Cell Signaling Technology #4782), rabbit anti-phospho-CREB (Ser133, 1:1,000, Millipore #06–519), rabbit anti-CREB (1:1,000, Millipore #AB3006), and mouse anti-α tubulin (1:10,000, GeneTex #GTX628802). Protein signals were visualized with horseradish peroxidase–conjugated secondary antibodies and ECL reagent (Thermo Fisher Scientific). Quantification of immunoblots was conducted with Image J software.

Mice and rats were killed by an overdose of urethane 24 h after dMCAO or craniectomy and then perfused with cold saline to remove circulating albumin from the blood. Isolated brains were coronally sectioned at 2 mm thickness. Motor cortex tissue from the middle two coronal sections (approximately +2 to +6 mm from λ), where infarct occurred or would have occurred, was isolated for Western blotting as described previously (Liao et al., 2019 (link)). In brief, these samples were homogenized in lysis buffer (20 mm Tris, 150 mm NaCl, 1 mm EDTA, and 1% NP-40 adjusted to pH 7.4) containing protease inhibitor (catalog #04693132001, Roche) and centrifuged at 12,000 rpm for 30 min. Proteins (∼30 μg) from the supernatants were heated for 5 min at 100°C, separated by electrophoresis, and transferred to PVDF membranes. After blocking in milk (5% dry milk powder in TBST) for 2 h at room temperature, each membrane was incubated with primary antibodies against α-tubulin (1:5000 in 2% BSA in TBST; catalog #GTX628802, Genetex) and albumin (1:5000 in 2% BSA in TBST; catalog #ab106582, Abcam) overnight at 4°C, washed with TBST, and incubated with goat anti-mouse IgG (1:1000 in 2% BSA in TBST; catalog #GTX213111-01, Genetex) and goat anti-chicken IgY (1:1000 in 2% BSA in TBST; catalog #ab97135, Abcam) secondary antibodies. Western blot images were taken using a ChemiDoc XRS+ System (BIO-RAD).

The astrocytes were fixed in −20° precooled acetone and methanol (1:1) for 5 min and then blocked with 5% bovine serum albumin (BSA) at room temperature for 30 min. The astrocytes were probed with the indicated primary antibodies (anti-TBCB, 1:50, A13248, ABclonal, China; anti-TBCB, 1:250, sc-377139, Santa Cruz, USA; anti-α- tubulin, 1:5000, GTX628802, GeneTex, Irvine, California, USA) properly diluted at 4° overnight. Then, the cells were incubated with secondary antibodies (FITC goat anti-rabbit IgG, 1:200, E031220-01, EARTH, China; Cy3 goat anti-mouse IgG, 1:200, Abbkine, Wuhan, China) and stained with DAPI (C1005, Beyotime, Shanghai, China). Subsequently, the cells were mounted in Fluorescence Mounting Medium (ab104135, Abcam, Cambridge, UK) and sealed with nail polish. Images were obtained by confocal laser scanning microscopy (Leica DMI8, Germany) and the intensity of the fluorescence was analysed by ImageJ (1.53 c) software (n = 6) [53 (link),55 (link)]. The astrocyte processes in the high magnification images were also counted by ImageJ (1.53 c) software (approximately 50 cells were counted in each group). IF was mainly used to observe the changes in cell morphology and protein distribution in this study.

Anti-ALDH2 (Proteintech, 15310-1-AP, 1:4000), anti-4-HNE (Abcam, ab46545, 1:2000), anti-APP C-Terminal Fragment (C1/6.1, Biolegend, 802801, 1:500), anti-β-amyloid (1–16) (6E10, Biolegend, 803001, 1:1000), anti-β-amyloid (1–40) (Cell Signaling Technology, 12990, 1:1000), anti-β-amyloid (1–42) (Cell Signaling Technology, 14974, 1:1000), anti-GM130 (Cell Signaling Technology, 12480S, 1:1000), anti-TGN46 (Invitrogen, MA3-063, 1:1000), anti-presenilin 1 (Sigma, MAB5232, 1:500), anti-PEN2 (Abcam, ab18189, 1:500), anti-APH1 (Invitrogen, PA1-2010, 1:1000), anti-Rab5 (Cell Signaling Technology, 3547, 1:1000), anti-RCAS1 (Cell Signaling Technology, 12290S, 1:1000), anti-VDAC (Cell Signaling Technology, 4661S, 1:1000), anti-LAMP2 (Proteintech, 27823-1-AP, 1:1000), anti-CANX (Cell Signaling Technology, 2679, 1:1000), anti-VPS35 (Abcam, ab10099, 1:1000), anti-LC3A (Cell Signaling Technology, 4599, 1:1000), anti-Iba1 (Wako, 019-19741, 1:500), anti-tau (Cell Signaling Technology, 46687, 1:1000), anti-phospho-S396-tau (Abcam, ab32057, 1:1000), anti-α-tubulin (GeneTex, GTX628802, 1:10,000), anti-β-actin (GeneTex, GTX124213, 1:10,000), anti-GAPDH (MBL, M171-3, 1:10,000), and anti-SorLA/SORL1 antibody (Abcam, ab190684, 1:1000).

Medium collected from cell culture in vitro (12 μl medium per culture) and tissue samples harvested from mice in vivo were denatured by boiling in lysis buffer, separated by electrophoresis, transferred onto PVDF membranes, and imaged after incubation in primary and secondary antibodies as described previously for in vitro [55 (link)] and in vivo [57 (link)] experiments. The following primary antibodies were used: anti-albumin (1:2000; GeneTex, cat. #102419, for the in vitro experiments; 1:5000; Abcam, cat. #ab106582, for the in vivo experiments) and anti-ɑ-tubulin (1:5000; GeneTex, cat. #GTX628802) antibodies, and the following secondary antibodies were used: anti-rabbit (1:5000; GeneTex, cat. #GTX213110-01), anti-mouse (1:1000; GeneTex, cat. #GTX213111-01), and anti-chicken (1:1000; Abcam, cat. #ab97135) antibodies.