Anti il 1β antibody

Cells were washed twice with ice-cold phosphate buffered saline (PBS) and then harvested and lysed in cold radioimmunoprecipitation assay (RIPA) buffer. Equal amounts of protein were separated by 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA). The membrane was blocked with 5% nonfat milk and incubated with anti-NLRP3 antibody, ASC antibody, anti-caspase-1 antibody, and anti-IL-1β antibody (Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Blots were developed using enhanced chemiluminescent substrate (Thermo Fischer Scientific Pierce, IL, USA).

In each group, five animals were sacrificed at T3, and the total protein was extracted from their hippocampal tissues. Protein levels of GABA_B1 receptors, NF-κB, IL-1β and TNFα were analyzed using western blot assay. Briefly, total protein concentrations were determined using bicinchoninic acid assays (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), and western blot assays were performed as described above. Primary antibodies included the anti-GABA_B1 receptor antibody (1:500, Santa Cruz Technology, Santa Cruz, CA), the anti-NF-κB p65 antibody (1:500, Santa Cruz Technology, Santa Cruz, CA), the anti-IL-1β antibody (1:500, Santa Cruz Technology, Santa Cruz, CA) and the anti-TNFα antibody (1:500, Santa Cruz Technology, Santa Cruz, CA), as well as the secondary horseradish peroxidase-labeled goat anti-rabbit immunoglobulin (1:4000, PTG Lab, Chicago, IL). The membrane was developed using enhanced chemiluminescence, exposed to X-ray film, and then photographed under UV light using a UVP gel documentation system (UVP, Upland, CA).

For western blotting analysis, tissue extracts were prepared by homogenizing mouse hippocampus tissue in T-PER Tissue Protein Extraction Reagent (Thermo Scientific Inc., Waltham, MA, USA) containing protease inhibitor, α-complete (Roche Applied Science, Penzberg, Germany). Fifteen micrograms of each tissue extract sample were adjusted to give a final solution of 60 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 0.1% bromophenol blue, and 5% β-mercaptoethanol. The solution was heated at 100ºC for 5 min, electrophoresed through a 10% SDS-polyacrylamide gel, and transferred to polyvinylidine difluoride membranes (Amersham Pharmacia Biotech, Buckinghamshire, UK). Plexin-A1, Neuropilin-1, COX-2, iNOS, IL-1β, TNF-α and β-actin were detected by their respective antibodies using an enhanced chemiluminescence western blot detection system (Amersham Pharmacia Biotech) according to the manufacturer’s instructions. Anti-Plexin-A1 antibody (Abcam), anti-Neuropilin-1 antibody (Abcam), anti-COX-2 antibody (Santa Cruz Biotechnology, Inc., Dallas, Texas, USA), anti-iNOS antibody (Merck Millipore, Darmstadt, Germany), anti-IL-1β antibody (Santa Cruz Biotechnology), anti-TNF-α antibody (Santa Cruz Biotechnology), and anti-β-actin antibody (Cell Signaling Technology, Danvers, MA, USA) were used.

To detect the expression of p-p38 in the 105 cases of GA tissues and in nude mice lung metastasic gatric cancer by immunohistochemistry (IHC), we used previously described methods [32 (link)-34 (link)], with the use of a specific anti-p-p38 antibody (#4631) (1:100 dilution, Cell Signaling Company, Danvers, MA, USA). The assessed standards for staining results were also the same as our previously described for p-Akt2 [33 (link)]. Statistical significance was analyzed by the Wilcoxon signed-rank test, Chi-square test, and the Fisher’s exact test. To assess the level of IL-1β, MMP-2 and 9, and c-fos in the tissues mention-above by IHC, we also used the same previous method [32 (link),33 (link)]. Anti-MMP-2 (ab110186) and MMP9 (ab38898), and c-fos (ab53036) antibodies used for IHC were 1:250, 1:200 and 1:200 dilution, respectively, and they were from Abcam (Cambridge, MA, USA); Anti-IL-1β antibody was from Santa Cruz (sc-7884) (Santa Cruz, CA, USA) and was diluted 1:100 before use. Spearman’s method was used to analyze the correlation in expression levels of p-p38 with IL-1β, MMP-2 and 9, and c-fos in GA tissue.

Dulbecco’s Modified Eagle Medium (DMEM), 1640 Medium and fetal bovine serum were obtained from Hyclone (Invitrogen Corporation, NY, USA), CCK-8 reagents were obtained from Dojindo (Kumamoto, Japan), oxidized low density lipoprotein (ox-LDL, MDA 30 μM) was obtained from Institute of Basic Medicine, Peking Union Medical College (Beijing, China), anti-LXRα antibody, anti-ABCA1 antibody were obtained from novusbio Biologicals (IL, USA), anti-CD68 antibody was obtained from Sigma-aldrich (CA, USA), anti-IL-1β antibody and anti-TNF-α antibody were obtained from Santa Cruz Biotechnology (TX, USA), PE-conjugated secondary antibodies and PE-conjugated secondary antibodies were obtained from Biolegend Inc (SD, USA),Oil Red O, atorvastatin, T-PER protein extraction reagent and SR9243 were obtained from Sigma-Aldrich (CA, USA), reporter genes were synthesized by Shanghai Genechem Co.,Ltd. (Shanghai, China), IL-1β and TNF-α ELISA kit were obtained from Dakewe Biotechnology (Beijing, China),BCA assay kit was obtained from Beyotime (Shanghai, China), bovine serum albumin was obtained from Sangon Biotech (Shanghai, China), HRP-conjugated anti-β-actin antibody was obtained from Kangchen Inc. (Shanghai, China).

Platelets (1–5 × 10⁶) were incubated with FITC-conjugated anti-CD41 (BD Phamingen) (1:20) and PE-conjugated anti-CD62-P (BD Pharmingen) (1:20) for 30 min at 37 °C. Phosphatidylserine exposure on platelets was measured by the binding of FITC-conjugated Annexin V (BD Pharmingen); mitochondrial membrane potential (Δ_Ψm) was measured using the probe tetramethylrhodamine ethyl ester (TMRE, Fluka Analytical) (100 nM, 10 min), active caspase-9 was determined using the probe FAM-LEDH-FMK green fluorescent inhibitor of caspase (FLICA, Immunochemistry Technologies), and platelet synthesis of nitric oxide (NO) was quantified using the probe DAF-FM diacetate (Invitrogen Molecular probes). For intracellular IL-1β labeling, isolated platelets were labeled with FITC-conjugated anti-CD41, fixed with 4% paraformaldehyde for 20 minutes, washed once, and permeabilized with Triton 0.1% for 10 minutes. Platelets were then incubated for 30 minutes with anti-IL-1β antibody (5 mg/mL; Santa Cruz Biotechnology), followed by incubation with secondary Alexa Fluor 546–conjugated anti-rabbit IgG for 30 minutes. Isotype-matched antibodies were used to control nonspecific binding of all antibodies. Platelets were distinguished by the expression of CD41 and characteristic forward and side scattering. A minimum of 10,000 gated events were acquired using a FACScalibur flow cytometer (BD Bioscience, CA).