Ma0186

We collected U251 cells and tissue samples and used RIPA lysis buffer (89900, Thermo Fisher Scientific, USA) to extract total proteins. Then measured them using a bicinchoninic acid (BCA) Protein Assay Kit (23227, Thermo Fisher Scientific, USA). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis separated the protein samples (n = 3), followed by their transfer onto polyvinylidene fluoride membranes. After 2 h of blocking in 5% non-fat milk, membranes were probed overnight at 4 °C with primary antibodies against FLAG (66008-4-Ig, proteintech, China, 1:5000), BAX (50599-2-Ig, proteintech, China, 1:2000), BCL-2 (68103-1-Ig, proteintech, China, 1:2000), GPX4 (ab125066 abcam, USA, 1:1000); SLC7A11 (DF12509, Affinity, USA, 1:1000), GAPDH (AF7021, Affinity, USA, 1:1000), and 2 h of secondary antibody (PR30011/PR30012, proteintech, China, 1:5000) incubation. Proteins were visualized by enhanced chemiluminescence (MA0186, Meilunbio, China) on a ChemiDoc camera (Bio-Rad), and protein expression levels were quantified using the ImageJ software (v1.8.0). Full and uncropped western blots are presented in Supplemental File.

Relative protein levels in whole brain samples were determined by WB analysis. The protein samples were separated and transferred to a polyvinylidene difluoride (PVDF) membrane. The PVDF membrane was incubated with primary antibodies at 4 °C overnight. The primary antibodies and their concentrations were as follows: NF-κB (1:1000, Cell Signaling, Cat# 8242), p-NF-κB (1:1000, Cell Signaling, Cat# 3033), MAPK (1:1000, Cell Signaling, Cat# 9212), p-MAPK (1:1000, Cell Signaling, Cat# 4631), CSF1R (1:100, SANTA CRUZ, Cat# 365719), IL-1β (1:1000, Abcam, Cat# Ab9722), TNF-α (1:1000, Cell Signaling, Cat# 11948), iNOS (1:2000, Proteintech, 18985–1-AP), Bcl-2 (1:1000, Proteintech, Cat# 26593–1-AP), Bax (1:2000, Proteintech, Cat# 50599–2-lg), Cle-caspase-3 (1:1000, Proteintech, Cat# 19677–1-AP), GAPDH (1:7000, Proteintech, Cat# 60004–1-lg), claudin-5 (1:500, Abcam, Cat# 15106), ZO-1 (1:1000, Proteintech, Cat# 21773–1-AP), and occludin (1:800, Proteintech, Cat# 66378–1-Ig). After being incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature for 2 h, the protein bands were visualized with an enhanced chemiluminescence kit (MA0186, Meilunbio). The protein bands were imaged with the Bio-Image Analysis system (Bio-Rad Laboratories, Inc.) and analyzed with ImageJ software.

The total protein was prepared by RIPA lysis buffer (R0010, Solarbio) and quantified by BCA assay (MA0082, Meilunbio, Dalian, China). Equal amounts of protein were separated on 10% SDS-PAGE and transferred to PVDF membranes. The membranes were incubated overnight at 4 °C with antibodies against cleaved-caspase 9 (1:2000, 9509s, CST, Shanghai, China), Bax (1:2000, TA810334, OriGene, Beijing, China), Bcl-2 (1:2000, UM870117, OriGene), GAPDH (1:8000, TA-08, ZSGB Bio, Beijing, China), phospho-ERK (1:1500, 4370T, CST), ERK (1:1500, 4695T, CST), phosphor-JNK (1:1500, 4668T, CST), JNK (1:1500, 9252T, CST), phosphor-p38 MAPK (1:1500, 4511T, CST) and p38 MAPK (1:1500, 8690T, CST), followed by incubation with HRP-conjugated secondary antibodies (1:8000, ZDR-5306 and ZDR-5307, ZSGB Bio). Protein bands were visualized by Meilunbio^® fg super sensitive ECL luminescence reagent (MA0186, Meilunbio) and imaged on a DNR MicroChemi chemiluminescence detection system (DNR, Israel). The protein bands were analyzed using ImageJ software.

Samples were gained from the supernatant after PMA stimulation as described above. In this experiment, the same amount of cells was seeded into each well of the 24-well plate, and the same volume of medium (200 μl) was used during the PMA stimulation. After treating with 80 nM PMA in each well for 1 h, the supernatant samples (150 μl) were loaded in 50 mmol/L Tris, pH 8.0, 1% SDS, 5% glycerol, and 0.002% bromophenol blue. The vWF multimer analysis was as previously described (Ma et al., 2016 (link)). Then, 1.2% agarose gels were prepared by dissolving Seakem high-gelling-temperature agarose (Lonza, 50,041) in 0.375 mol/L Tris (pH 8.8), with SDS added to a final concentration of 0.1%. The gels were run at 30 V for 16 h (Tanon, EPS600) before transferring to a nitrocellulose membrane labeled with rabbit anti-vWF antibody (1:10,000), followed by horseradish peroxidase-conjugated anti-rabbit secondary antibody (1:5,000), and developed by chemiluminescence (Meilunbio, MA0186). The multimers of each lane on the same gel were arranged according to their molecular weight. The multimer gels were analyzed using the NIH ImageJ software. The quantification of supernatant vWF multimers was carried out based on the normalization of the β-actin protein level of the cells in each well.