β tubulin

The whole hippocampus (ventral and dorsal) was lysed in RIPA buffer containing protease inhibitors and phosphatase inhibitors. Protein concentration was determined colorimetrically by NANODROP 2000 (Thermo Scientific). Protein lysates were separated by 10% SDS-PAGE electrophoresis and were transferred onto polyvinylidenedifluoride (PVDF) membranes. After blocking with 5% BSA for 1 hr, and the following antibodies were used: NR1(Santa Cruz Biotechnology, 5704S, 1 : 500), AKT/p-AKT (Cell Signaling Technology, 9272S, 4058s, 1:500), mTOR/p-mTOR (Cell Signaling Technology, 2972S, 2971S,1:500), p70S6 kinase p70s6k) / p--p70S6K (Cell Signaling Technology, 2708S, 9234S, 1:500), p-4EBP1 (Cell Signaling Technology, 9644S.1:1000), GluR1 (Cell Signaling Technology, 12551S, 1:1000) and β-tubulin (Epitomics, 1879-1, 1:2000). After the blots were incubated with antibodies overnight at 4 °C, they were incubated with horseradish peroxidase- conjugated secondary antibodies for 1 hr. The blots were visualized using the SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific Inc.). The resulting data was analyzed statistically using SPSS Statistics 20. All experiments were performed in troplicate. The final data are expressed as a ratio of the relative optical density (ROD) of the protein of interest to the ROD of β-tubulin. A p-value < 0.05 was considered significant.

Lipopolysaccharide was purchased from Sigma-Aldrich (St Louis, Ca). Recombinant mouse IL-4, recombinant mouse IL-6, recombinant mouse IL-1β, recombinant mouse TNF-α, recombinant mouse IL-10, and recombinant mouse TGF-β were purchased from R&D Systems (Minneapolis, MN). Tocilizumab, LY2109761, R-7050, Stattic, Bay 11-7082, and SIS3HCl were purchased from Selleck (Huston, USA). IL-1 receptor antagonist (IL1Ra) was purchased from Make Research Easy (Nanjing, China). Monoclonal mouse anti-β-actin (TA-09) was purchased from Zhongshan Golden Bridge Biotechnology (Beijing, China). Antibodies against OT, OTR, β-tubulin, CD206, F4-80, phospho-STAT3, STAT3, phospho-SMAD2, SMAD2, phospho-SMAD3, SMAD3, p65, and phospho-p65 were purchased from Abcam (Cambridge, UK) and Cell Signaling Technology (Danvers, MA). Secondary antibodies were purchased from Invitrogen Life Technology (Foster City, CA). Mouse ELISA kits were obtained from R&D Systems and CUSABIO (Wuhan, China). All reagents were analytical grade.

Nuclear and cytoplasmic protein was extracted from cells by suspension in a solution of Buffer A containing 10 mM HEPES pH 7.4, 1.5 mM MgCl₂, 10 mM NaCl, 0.1% NP-40 and a cocktail of protease inhibitors (Complete Mini Cocktail, Roche Diagnostics Ltd, Lewes, UK). The cells were lysed on ice for 10 min after which they were passed through a 21 gauge needle to ensure complete plasma membrane lysis. Nuclei were pelleted by centrifugation (12,000 x g at 4°C for 2 min) and the supernatant containing cytoplasmic protein was retained. Nuclei were washed in a solution of Buffer A (as specified above). The nuclei were lysed for 10 min on ice in Buffer A and sonicated for 30 sec to completely disrupt the nuclear membrane. Remaining cell debris was pelleted by centrifugation and the supernatant containing nuclear protein retained. Total protein content was determined by the Bradford protein method using the BCA protein assay kit (Pierce, Cramlington, UK). Protein (30 μg) was loaded onto a Tris-Glycine polyacrylamide gel (10%) and subsequently transferred to a nitrocellulose membrane. The antibodies used for Western blotting were β-tubulin (Abcam 1:1000), HOXD10 (Biorbyt 1:200), POU2F1 (Abcam 1:1000) and TATA-BP (Abcam 1:1000).