Las 4000 imaging system

The total protein content was extracted from the penumbral area of the ischemic hemispheres and BV2 cells. Brain tissue in the ischemic hemisphere was homogenized with lysis buffer (50 nM/L NaCl, 1 mM/L EDTA, 1% Triton X-100, 0.5% SDS, 0.5% sodium deoxycholate and 20 mM/L Tris HCl; pH 7.5) and centrifuged at 15,000× g for 20 min. Protein samples (50 μg per lane) were run on a polyacrylamide gel, transferred to a PVDF membrane (Millipore, Billeria), and blocked with a 5% milk solution (nonfat dry milk in PBST) for 2 h. Then, the membranes were incubated with the primary antibodies against TNF-α, IL-1β, IL-6, Arg1 and YM1 and β-actin (1:1000, all from Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C. After washing with TBST 3 times, and the membranes were then incubated with the corresponding conjugated anti-rabbit IgG (1:10,000; Cell Signaling Technology) at room temperature for 2 h. Immunoreactivity was detected by an enhanced chemiluminescence (ECL) kit (Millipore, Billeria), and the relative densities of the protein bands were scanned using a LAS 4000 Fujifilm imaging system (Fujifilm, Tokyo, Japan) and analyzed by densitometric evaluation using Quantity-One software Version 4.6.6 (Bio-Rad Hercules, CA, USA).

Proteins were extracted from hippocampal tissue using RIPA buffer containing proteinase (Complete, Roche) and phosphatase inhibitors (1 mM β-glycerophosphate, 10 mM NaF, and 0.1 mM Na₃VO₄). Total protein was determined using a BCA protein assay kit, run on polyacrylamide gel, and transferred on to a PVDF membrane (Millipore, Billeria). The membrane was blocked with 10% milk in PBS for 1 h at room temperature and incubated overnight at 4 °C with synaptophysin (SYP) (1:1000, cell signal technology) and anti-α-tublin (1:1000, Abcam) antibodies. After washing five times with TBST, the membrane was incubated with anti-rabbit IgG-HRP secondary antibody (1:10000, cell signal technology) at room temperature for 1 h. Proteins were detected with a chemiluminescence (ECL) kit (Millipore, Billeria). Then, the relative density of protein bands was scanned using an LAS 4000 Fujifilm imaging system (Fujifilm, Tokyo, Japan) and analyzed by Quantity-One software (Bio-Rad Hercules, CA, USA).

The brain tissues were homogenized thoroughly in a RIPA lysis buffer containing 150 mM NaCl, 50 mM Tris (pH 7.4), 1% NP40, 0.5% sodium deoxycholate, and 0.1% SDS. Then, the samples were centrifuged at 25,000 g at 4°C for 60 min, and the supernatants were collected and stored at –80°C until use. The serum samples from these subjects were also dissolved in the above buffer. Before that, protein concentration was measured with a BCA kit. Subsequently, western blot analysis was made according to the protocol previously published. All the samples were subjected to electrophoresis, transferred onto PVDF membranes, and incubated with the primary antibodies: rabbit anti-Alix (1:500, Cell Signaling Technology), mouse anti-β-actin (1:10,000, Sigma), and mouse anti-IgG (1:10,000, Abcam). Digital images were obtained with the LAS4000 FujiFilm imaging system (FujiFilm, Japan), and the densitometric analysis was made by Quantity-One software (Bio-Rad, United States).

Total cellular protein extraction and Western blotting were performed as previously described [69 (link)]. For each target protein, lysate samples with equal protein amounts (10–50 µg total) were incubated overnight at 4 °C with the primary antibodies listed in Table 1. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies, goat anti-mouse IgG (Invitrogen), or goat anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA, USA). The blots were developed using Immobilon-Western chemiluminescent HRP substrate (Millipore Corp., Billerica, MA, USA) and visualized using the LAS4000 imaging system (Fujifilm Corp., Tokyo, Japan) or Amersham ImageQuant™ 800 systems (Cytiva Life Sciences, formerly GE Healthcare Life Sciences, MA, USA). The relative intensities of the protein bands were quantified using ImageJ 1.51v software (NIH).

We used monoclonal antibodies raised against GFP (clones 7.1 and 13.1; Roche Diagnostics, Meylan, France), HA (clone F7; Santa Cruz Biotechnology, Dallas, TX), anti-ubiquitin antibody coupled to horseradish peroxidase (clone P4D1; Santa Cruz Biotechnology), and polyclonal antibodies against 3-phosphoglycerate kinase (PGK) (clone 22CS; Life Technologies, Saint Aubin, France). Immunoblots were acquired with the LAS-4000 imaging system (Fuji, Tokyo, Japan). Quantification was performed using ImageJ (NIH) on non-saturated blots.

After cell transfection, proteins were extracted using RIPA buffer (Beyotime, Shanghai, China) and quantified using a bicinchoninic acid (BCA) protein assay kit (Beyotime, Shanghai, China). Then, 30 µg/lane protein was separated via SDS-PAGE and transferred to PVDF membranes. The PVDF membranes were blocked at room temperature and then incubated overnight at 4 °C with rabbit-anti-human SALL1 (1:1,000; cat.no.ab41974; Abcam, Cambridge, MA, United States) and GAPDH (1:1,000; cat.no.ab8245; Abcam, Cambridge, MA, United States) primary antibodies. The next day, membranes were incubated with a specific secondary antibody conjugated to horseradish peroxidase for 2 h at room temperature, and then the signals were detected using an enhanced chemiluminescence system kit (Pierce, Waltham, MA, United States) and visualized using an LAS-4000 imaging system (Fujifilm Holdings Corporation, Tokyo, Japan).