Anti tnf α

Total proteins from the ischemic cortical area brain tissues or BV2 microglial cells were collected in equal amounts of cell lysate, and the separated proteins in the supernatant were subsequently transferred. For immunoblotting, the following primary antibodies were used: Anti-Dectin-1 (1:1,000; Abcam), anti-Syk (1:1,000; Cell Signaling Technology, Inc.), anti-p-Syk (1:1,000; Cell Signaling Technology, Inc.), anti-TNF-α (1:1,000; Cell Signaling Technology, Inc.), and anti-iNOS (1:1,000; Cell Signaling Technology, Inc.). Western blotting was performed at 6 h and 3, 5, and 7 days after ischemic stroke for the mouse tissues and at 0, 3, 6, and 12 h after OGD/R treatment and 3, 6, 12, and 24 h after LPS treatment for the BV2 microglial cells. Then, the optimal time points of 3 days for the in vivo experiments and 3 h for OGD/R treatment and 24 h for LPS treatment in vitro were selected for the subsequent experiments.

Hepatic and pancreatic tissue lysates from control and STZ-induced male and female rats containing equal amounts of protein were separated by SDS-PAGE. After electrophoresis, proteins were transferred to polyvinylidene difluoride (PVDF) membranes, blocked for 1h with 5% skim milk at room temperature, and incubated with the indicated primary antibodies for 2 h at 1:1000 dilution (anti-β-actin, anti-SPARC, anti-C/EBPβ, anti-ATP5B, anti-CD95, anti-MCP1, anti-iNOS, anti-Cyt C, anti-SOD2, anti-INS, anti-CA3, anti-RARhoGAP, anti-CPS1, anti-BHMT, anti-PA, anti-APC2 [Santa Cruz Biotechnology, Santa Cruz, CA, USA], anti-TNFα, anti-PARP-1, anti-NFκB, anti-HSP90 [Cell Signaling Technology, Beverly, MA, USA], and anti-CRP [AbFrontier, Seoul, Korea]). After washing with Tris-buffered saline containing Tween 20, the membranes were incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. Then, immune complexes were detected using the ECL method, and immunoreactive bands were quantified by densitometric analysis using ImageMaster 2D software version 4.95 (GE Healthcare, Little Chalfont, Buckinghamshire, UK). Relative intensity (%) values of proteins were normalized to β-actin levels.

For protein analysis of proinflammatory cytokines in the para-hematoma tissues, the animals was euthanized via intracardial perfusion of PBS and the brain tissues around hematoma were collected. Proteins obtained from six rats per group were extracted in a buffer (1% Triton X-100 with 1 mg/ml leupeptin, 1 mM PMSF, and 1 µg/ml pepstatin; pH 7.4) and centrifuged at 12,000 g for 30 min at 4 °C. Equivalent protein amounts (20 μg) were loaded in each lane of SDS-PAGE gels. Following gel electrophoresis, the protein ladders were transferred to a PVDF membrane (Millipore, US), blocked with 10% bovine serum albumin (BSA), and incubated in primary antibodies and the corresponding secondary antibodies. The following antibodies were used: anti-IL1β (1:1000; R&D System, US) and anti-TNF-α (1:1000; Cell Signaling technology, US). The immunoreactive bands were visualized with an ECL kit (Amersham Biosciences, Arlington Heights, IL) following the manufacturer’s instructions. The data were analyzed via densitometry with ImageJ software. β-actin was used as an internal loading control (1:1000; Santa Cruz, US).

Total proteins were extracted from liver tissue using a protein extraction kit (Minute Protein Extraction Kit; Invent Biotechnologies, Inc., Eden Prairie, MN, USA). Extracted proteins were quantitated by the BCA method (BCA protein Assay Kit; Takara Bio, Inc., Otsu, Japan), and were diluted to the same protein concentrations. Equivalent amounts (50 µg) of each group were run on SDS polyacrylamide gels. Proteins were transferred electrically to a PVDF membrane and incubated with the following antibodies; anti-TNF-α, anti-IL1β (Cell Signaling Technology, Inc., Beverly, MA, USA), anti-DMT1, anti-hepcidine25, anti-CPT1A, anti-CPT2, anti-SOD2, anti-catalase (Abcam, Tokyo, Japan), anti-GPx4 (LifeSpan BioSciences, Seattle, WA, USA). Membranes were washed and incubated with respective secondary antibodies. Detection of bands was performed by chemiluminescent analysis. Beta actin was used as a loading control. Representative data of three experiments were shown on figures.

The total protein from pancreatic tissue and isolated PACs was extracted in PRO-PREP Protein Extraction Solution (Intron Biotechnology) on ice according to the manufacturer’s instructions, and the protein concentrations were measured using the Bio-Rad DC Protein Assay Kit (Bio-Rad, Hercules, CA, USA). A total of 20 μg of each of the proteins was subjected to electrophoresis on 10% sodium dodecyl sulphate-polyacrylamide gels, and the protein bands were transferred onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were incubated in 5% non-fat dry milk in Tris-buffered saline containing 0.1% Tween-20 for 1 h and treated with antibodies against anti-caspase-12 (1:500), anti-CCAAT-enhancer-binding protein homologous protein (CHOP) (1:500), anti-78-kDa glucose-regulated protein (Grp78) (1:1000) (all from Cusabio Biotech Co), anti-TNF-α (1:1000), anti-NF-κB-p65 (1:1000), and anti-NF-κB-p-p65 (1:1000) (Cell Signaling Technology, Beverly, Massachusetts, USA) at 4 °C overnight. The membranes were incubated with anti-rabbit or anti-mouse IgG (Santa Cruz Biotechnology) as the secondary antibodies (1:2000) for 1 h. The protein bands were visualised using enhanced chemiluminescence (Advansta, Menlo Park, CA, USA) and normalised to the levels of β-actin (Santa Cruz Biotechnology).

For immunoblotting analysis, lung samples and cell lysates lysed by RIPA buffer (Beyotime, China) were resolved by SDS–PAGE and transferred to the polyvinylidene fluoride membrane (Millipore, United States). Immunoblots were visualized by the ECL system (Fdbio Science, China). The following antibodies were used in immunoblotting analysis: antibodies for anti-IFITM3 (1:2,000), anti-MFN2 (1:2,000), anti-IFN-β (1:500) antibody and anti-IL-1β (1:1,000) were from Abcam. β-actin antibody (1:2,000) was purchased from Fude Biotechnology. Anti-phospho-IRF3 (1:1,000), and anti-TNF-α (1:2,000) antibodies were purchased from Cell Signaling Technology. Anti-MAVS (1:500) antibodies were obtained from Proteintech Group. β-actin was used as an internal control and the relative densities of the measured protein were quantified by image J software.