P nf κb

After centrifugation (10,000 g, 15 min at 4°C), the supernatants were collected for protein analysis. The protein concentration was determined by Bradford protein assay. Western blot analysis was performed as previously described. Briefly, 50 µg proteins from each sample were loaded onto an SDS polyacrylamide gel for electrophoresis and subsequently transferred to polyvinylidene fluoride membranes. The membranes were blocked with 5% nonfat dry milk in PBS for an hour and incubated with a primary antibody against BMAL1 (1:200 dilution), AKT (1:1000 dilution), p-AKT (Thr 308) (1:2000 dilution), mTORC1 (1:1000 dilution), p-mTORC1 (Ser2481) (1:2000 dilution), S6K1 (1:1000 dilution), p70-S6K1 (Thr389) (1:2000 dilution), NfκBp65 (dilution 1:250 dilution), p-NfκB (Ser536) (1:250 dilution) overnight at 4°C (BMAL1, mTORC1, p-mTORC1, NfκBp65 and p-NfκB were purchased from Abcam; AKT, p-AKT, S6K1S and p70-S6K1 were purchase from CST Danvers, USA). Samples were then incubated for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz, USA) (1:5000 dilution). Band intensities were visualized with chemiluminescence reagent (Millipore Corp.) by the BioMax film (Kodak) and then analyzed with Gel-Pro analyzer 4.0 software. β-Actin (Santa Cruz, USA) protein was utilized as a loading control.

Proteins were extracted from cells and 30 µg samples were fractionated by SDS-PAGE then transferred to nitrocellulose membranes. Membranes were blocked with 5% BSA in TBS-T (TBS with 0.05% Tween 20) and incubated with primary antibodies against p-Akt(473), Akt, p-Foxo3a, Foxo3a, raptor(1:1,000 dilution, Cell Signaling Technology, Danvers, MA, USA); Actin, RANKL, M-CSF, OPG,p16,p19,p27,p21,cdk2,p-NF-κB, NF-κB,p-STAT3, STAT3, α-SMA, and E-cadherin(1:1,000 dilution, Abcam).

Raltegravir (purity >98%; No. 518048-05-0) and MCC950 (purity >99%, an NLRP3 inhibitor; No. 210826-40-7) were purchased from Hanxiang Biomedical Company (China). Rabbit polyclonal antibodies specific for NLRP3, high mobility group box 1 (HMGB1), toll-like receptor 4 (TLR4), phosphorylated nuclear factor-κB (p-NF-κB), claudin 18.1, and aquaporin 5 were obtained from Abcam Biotechnology (China). Anti-vascular endothelial cadherin (VE-cadherin) was purchased from Cell Signaling Technology (China). BAY11-7082 was from Beyotime (China). Fluorescein isothiocyanate (FITC)-labeled dextran was purchased from Xiao You Biotechnology (China). A myeloperoxidase (MPO) ELISA kit was purchased from the Bio-Swamp Biological Technology Company (China).

The spinal cord tissues were taken out from liquid nitrogen, lysed in RIPA lysis buffer, containing protease inhibitor, for 30 min and centrifuged to obtain the supernatant which was used to determine the protein concentration. After SDS-PAGE, the proteins were transferred onto a membrane, sealed and incubated overnight at 4°C with antibodies against NF-κB (Abcam, USA), p-NF-κB (Abcam, USA), B-cell lymphoma-2 (Bcl-2) (Abcam, USA), Bcl-2-associated X protein (Bax) (Abcam, USA), phosphatidylinositol 3-kinase (PI3K) (Abcam, USA), protein kinase B (Akt) (Abcam, USA), phosphorylated (p)-Akt (Abcam, USA), heme oxygenase-1 (HO-1) (Abcam, USA), Nrf2 (Abcam, USA), trithorax-1 (TRX-1) (Abcam, USA), Raf-1 (Abcam, USA), MEK (Abcam, USA), ERK (Abcam, USA), p-MEK (Abcam, USA), p-ERK (Abcam, USA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Abcam, USA) (diluted at 1:1000). Next, the PVDF membrane (Sigma, USA) was cleansed in TBST and incubated with horseradish peroxidase-labeled secondary antibodies at room temperature for 2 h. Finally, the color of the proteins was developed using an ECL kit and gel imaging system, and the absorbance analyzed by Image Tools.

Cells were lysed in RIPA buffer (50 mM Tris-Cl at pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA, 1 μg/mL leupeptin, 1 μg/mL aprotinin, 0.2 mM PMSF). Proteins (20–40 µg) were separated on 8–10% SDS/PAGE gel and then transferred onto PVDF membranes (Millipore, IPVH00010). After the blocking procedure, membranes were incubated with primary antibodies (1:1000) and HRP-conjugated secondary antibodies (1:5000), and visualized in Imager (Bio-Rad) using ECL system (Thermo Fisher Scientific, 34095). Antibodies used were: PD-L1 (R&D, MAB1086), NKG2D (R&D, MAB139), PD-1 (R&D, MAB1086), p-JAK1 (Y1022, Assay Biotech, A7125), p-JAK2 (Y1007 + 1008, Abbomax, 601–670), JAK1 (Abgent, AP20699a), JAK2 (Abgent, AP20700c), p-Stat1 (S727, Millipore, 07–714), Stat1 (Abgent, AP19835Bb), p-Stat3 (Y705, Abcam, ab76315), Stat3 (Abcam, ab5073), p-Stat5 (Y694, Abcam, ab32364), Stat5 (Abcam, ab16276), p-MAPK (Cell Signaling, 9101 S), p-Erk (Cell Signaling, 4695), p-Akt (S473, Cell Signaling, 9271), p-NFκB (S536, Abcam ab86299), and GAPDH (Cell Signaling, 2118 S).

Cells were lysed in RIPA buffer (50 mM Tris-Cl at pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA, 1 μg/mL leupeptin, 1 μg/mL aprotinin, 0.2 mM PMSF). Proteins (20-40 μg) were separated on 8–10% SDS/PAGE gel and then transferred onto PVDF membranes (Millipore, Billerica, MA). After blocking procedure, membranes were incubated with primary antibodies (1:1000), HRP-conjugated secondary antibodies (1:5000), and visualized in Imager (Bio-Rad) using ECL system (Thermo Fisher Scientific, 34095). Antibodies used were; PD-L1 (R&D, MAB1086), p-JAK1 (pY1022, Assay Biotech, A7125), p-JAK2 (pY1007 + 1008, Abbomax, 601-670), p-MAPK (cell Signaling, 9101S), p-Erk (Cell Signaling, 4695), p-MEK (Ser 217/221, Cell Signaling, 9121), p-Akt (S473, Cell Signaling, 9271), and p-Stat3 (pY705, Abcam, ab76315), p-Stat1 (pY701, Abbomax, 620-160), p-Stat5 (Abcam, pY694, ab32364), p-NFκB (S536, Abcam ab86299), p-mTOR (Millipore, 09-345), JAK1 (Abgent, AP20699a), JAK2 (Abgent, AP20700c), Stat1 (Abgent, AP19835b), MEK (Ameritech, ATB-T2715), MAPK (Signalway, 21245), ERK (Ameritech, ATB-T5371), Akt (Santa Cruz, sc5298) and GAPDH (Cell Signaling, 2118S).