Proteins from vascular tissue and cultured VSMCs were lysed by protein lysis buffer. Proteins were run on 8–10% SDS-PAGE to separation and then electro-transferred to PVDF membranes (Millipore). The membranes were blocked with 5% milk in TTBS for 2 h at room temperature and incubated overnight at 4 °C using the primary following antibodies: anti-IL-1β (1:1000, Proteintech, 16,806–1-AP), anti-IL-6 (1:1000, Proteintech, 21,865–1-AP), anti-TNF-α (1:1000, Proteintech, 6029–1-Ig), anti-PTEN (1:1000, Abcam, ab32199), anti-pan-AKT (1:1000, Abcam, ab8805), anti-pan-AKT (phospho T308; 1:1000, Abcam, ab38449), and 1:1000 anti-β-actin (1:2000, Santa, sc-47778). In the next day, after washing in the TTBS for three times, membranes were incubated with a 1:5000 dilution of anti-rabbit or anti-mouse antibody (Santa Cruz) for 1 h at room temperature. Protein bands were detected by enhanced chemiluminescence (ECL) Fuazon Fx (Vilber Lourmat).
Anti il 6
Manufactured by Proteintech
Sourced in United States, China
Anti-IL-6 is a laboratory reagent used for the detection and quantification of interleukin-6 (IL-6) in various sample types. It functions as a specific antibody that binds to IL-6, enabling its measurement through immunoassay techniques.
Automatically generated - may contain errors
Lab products found in correlation
Anti il 1β, by Proteintech (9 mentions)
Anti tnf α, by Proteintech (7 mentions)
Anti gapdh, by Proteintech (5 mentions)
Anti nf κb, by Cell Signaling Technology (2 mentions)
Anti jnk, by Cell Signaling Technology (2 mentions)
Anti β actin, by Proteintech (2 mentions)
Anti cox2, by Proteintech (2 mentions)
Anti tlr4, by Santa Cruz Biotechnology (2 mentions)
Ecl assay kit, by Yeasen (2 mentions)
Ripa buffer, by Beyotime (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Ab32199, by Abcam (1 mentions)
Ab8805, by Abcam (1 mentions)
Anti pan akt, by Abcam (1 mentions)
Anti rabbit or anti mouse antibody, by Santa Cruz Biotechnology (1 mentions)
Fuazon fx, by Vilber (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Cell lysis buffer, by Cell Signaling Technology (1 mentions)
Anti vimentin 60330 1 ig, by Proteintech (1 mentions)
27 protocols using anti il 6
1
Western Blot Analysis of Vascular Protein Markers
2
Western Blot Analysis of Protein Expression
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Total protein was extracted from cell lysates using cell lysis buffer (Cell Signaling Technology). The protein concentration was measured using the BCA Protein Assay kit (Solarbio). Then, protein was loaded into SDS- PAGE gels and transferred onto PVDF membranes, and blocked with rapid Closure Solution (WB1601, Biotides) for 20min. The membranes were incubated with primary antibodies including anti-IL6 (77888-1-Ig, Proteintech), anti-N-cadherin (22018-1-AP, Proteintech), anti-Vimentin (60330-1-Ig, Proteintech), anti-STAT3 (10253-2-AP, Proteintech), anti-GAPDH (60004-1-Ig, Proteintech) and anti-β-actin (ab8226, Abcam) at 4 °C overnight. The next day, the secondary antibodies were added onto the membranes at 37 °C for 1 h. Protein bands were visualized using the Image Lab Software (Bio-Rad).
3
Western Blotting for Liver Protein Expression
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The protein expression in the liver was determined through Western blotting following a previously described protocol [20 (link)]. The membranes were then incubated with primary antibody (PPARγ, IL-6, IL-1β, SIRT1, LXRα, pAMPKα, AMPKα, PPARα, pACC, ACC, FAS, SREBP1c, CPT1B, and β-actin) at room temperature for 2 h. In this study, the primary antibodies were anti-PPARγ (Cat# 2435S, 1:1000, Cell Signaling, Danvers, MA, USA), anti-IL-6 (Cat# 21865-1-AP, 1:1000, Proteintech, Rosemont, IL, USA), anti-IL-1β (Cat# 16806-1-AP, 1:1000, Proteintech), anti-SIRT1 (Cat# 9475S, 1:1000, Cell Signaling), anti-LXRα (Cat# ab176323, 1:1000, Abcam, Cambridge, UK), anti-pAMPKα (Cat# AF3423, 1:1000, Affinity, San Francisco, CA, USA), anti-AMPKα (Cat# AF6423, 1:1000, Affinity), anti-PPARα (Cat# sc-398394, 1:500, Santa Cruz, Santa Cruz, CA, USA), anti-pACC (Cat# D7D11,1:2000, Cell Signaling), anti-ACC (Cat# C83B10, 1:2000, Cell Signaling), anti-FAS (Cat# C20G5, 1:2000, Cell Signaling), anti-SREBP1c (Cat# ab28481, 1:2000, Abcam), anti-CPT1B(Cat# DF3904, 1:500, Affinity), and anti-β-actin (Cat# GTX109639, 1:10000, Genentech, San Francisco, CA, USA). The relative expression of proteins was quantified densitometrically using ImageJ software (Wayne Rasband, Madison, WI, USA), and β-actin was used as the internal control.
4
Inflammatory Cytokine Signaling Modulation
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N-acetylcysteine (NAC) was purchased from Sigma-Aldrich (St. Louis, MO, USA). The following antibodies were used in this research: anti-TNF-α (0.1 ug/ml; Santa Cruz, CA, USA), anti-IL-6 and anti-IL-2 (both from Proteintech, CA, and USA), anti-Bax, anti-Bcl-2, anti-Beclin 1, anti-LC3, anti-JNK, anti-p-JNK, anti-NF-κB (all from Cell Signal Technology, USA). ROS Fluorescent Probe-DHE was purchased from Vigorous. (Beijing, China).
5
Western Blot Analysis of Neuroinflammatory Markers
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Total protein from PC12 cells or the hippocampal tissues was extracted and analyzed using Western blots. Prepared samples were separated by 10% or 12% SDS-PAGE and transferred to Polyvinylidene Fluoride (PVDF) membranes (Millpore, Bedford, MA, USA). The membranes were incubated with anti-GAPDH (Abcam, Cambridge, UK), anti-thr181-phosphorylated-tau, anti-thr205-phosphorylated-tau, anti-ser396-phosphorylated-tau, anti-total tau, anti-JNK, anti-phospho JNK, anti-ERK1/2, anti-phospho ERK1/2, anti-phospho p38, anti-p38, anti-NF-κB, anti-phospho NF-κB (CST), anti-IL-6, and anti-TNF-α (Proteintech, Wuhan, China) antibodies. Immunoreactive bands were detected with the appropriate horseradish peroxidase-conjugated secondary antibodies and immunological complexes were visualized by enhanced chemiluminescence reagents (Pierce, Rockford, IL, USA).
6
Western Blot Analysis of Inflammatory Markers
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Protein concentration was detected using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) in accordance with the manufacturer's instructions. Total proteins were electrophoretically separated on sodium dodecyl sulfate polyacrylamide gels (8–15%) according to the molecular size of the target protein and were subsequently transferred onto polyvinylidene difluoride membranes. After being blocked with 5% skim milk, the membranes were incubated at 4°C overnight with the following primary antibodies: anti-RANKL, anti-OPG, anti-IL-1β, anti-IL-6, anti-TNF-α, and anti-β-actin (Proteintech, Chicago, IL, USA).
Then, the membranes were washed thoroughly and incubated with secondary antibodies (1 : 2000 anti-mouse/rabbit, Santa Cruz Biotechnology, Dallas, TX, USA) at room temperature for 2 hours. The signals were visualized using the enhanced chemiluminescence method (Immobilon Western Chemiluminescent HRP Substrate, MilliporeSigma). The samples were analyzed in duplicates, and the experiment was performed three times.
Then, the membranes were washed thoroughly and incubated with secondary antibodies (1 : 2000 anti-mouse/rabbit, Santa Cruz Biotechnology, Dallas, TX, USA) at room temperature for 2 hours. The signals were visualized using the enhanced chemiluminescence method (Immobilon Western Chemiluminescent HRP Substrate, MilliporeSigma). The samples were analyzed in duplicates, and the experiment was performed three times.
7
Western Blot Analysis of Inflammatory Markers
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Radio‐immune precipitation assay lysis buffer (Beyotime, P0013B) was used for tissue and cell lysates. BCA assay kit (Beyotime, P0012S) was used for protein concentration determination. After 30 µg protein amount was separated using 4%–20% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, it was transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% skim milk at 37°C for 2 h and then incubated with primary antibodies at 4°C overnight and secondary antibodies at 37°C for 1 h. After washing, the membranes were visualized by an ECL Kit (MedChemExpres) and quantified with ImageJ software. All experiments were repeated in triplicate for statistics. Following primary antibodies were used: anti‐iNOS (1:1000, Proteintech), anti‐COX‐2 (1:1000, Proteintech), anti‐IL‐6 (1:1000, Proteintech), anti‐TLR4 (1:1000, Proteintech), anti‐MYD88 (1:1000, Proteintech), anti‐NF‐κB‐p65 (1:1000, Affinity), anti‐NF‐κB‐p‐p65 (1:1000, Affinity), anti‐CD74 (1:1000, Abcam), β‐Actin (1:2000, Abcam).
8
DMSO, L-THP, and ConA Hepatoprotective Protocol
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Dimethyl sulfoxide (DMSO), L-THP, and ConA were purchased from Sigma-Aldrich (St. Louis, MO, USA). ALT and AST microplate test kits were purchased from Jiancheng Bioengineering Institute (Nanjing, China). Enzyme-linked immunosorbent assay (ELISA) kits for TNF-α and IL-6 were purchased from Anogen-Yes Biotech (Mississauga, Canada). The antibodies used in the study were from Cell Signaling Technology (Danvers, MA, USA) and included anti-IL-6, -LC3, -p-JNK, -TNF-α, -β-actin, -Bax, -Bcl-2, -cleaved caspase 3, -cleaved caspase 9, -Beclin-1, -TRAF6, and -JNK from Proteintech (Chicago, IL, USA). PCR kits were purchased from Takara (Dalian, China). RPMI-1640 medium was purchased from Thermo (Shanghai, China). The Pan T cell isolation kit was purchased from Miltenyi Biotec (Bergisch Gladbach, Germany).
9
Western Blot Analysis of Neuroinflammation
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We carried out the western blot analysis based on the standard procedure in previous reports. The dorsal root ganglion tissues were homogenized in cold RIPA lysis buffer with protease inhibitors. After centrifugation at 10,000 g for 20 min, the supernatant was collected for protein quantification. 10 μg of protein was separated by 10% SDS-PAGE and transferred onto the PVDF membrane (Roche). Then, the blot was blocked with defatting milk powder (5%) at room temperature for one hour. After that, the blot was incubated overnight with the following primary antibodies: anti-IL-6 (1 : 1000, Proteintech, USA), anti-MAPK1 (1 : 200, Proteintech, USA), anti-TP53 (1 : 200, Proteintech, USA), and anti-GAPDH (1 : 1000, Proteintech, USA). After incubation, the blots were incubated with horseradish peroxidase-labeled secondary antibody for 60 min at room temperature. Following incubation, the blots were measured by a chemiluminescence reagent (PerkinElmer, USA), and the band intensity quantification was performed using ImageJ software (NIH). GAPDH was used as an endogenous control.
10
Immunohistochemical Analysis of Inflammatory Markers
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4 μm paraffin sections were used for immunohistochemistry, and following the elimination of the endogenous peroxidase activity and antigen reparation, the tissues were sealed with blocking buffer. Then, the tissues were incubated with anti-TNF-α, anti-IL-6, and anti-IL-1β (Proteintech Group, Shanghai, China) overnight at 4°C and subsequently with the secondary antibody. The DAB chromogen was used for incubation, and then hematoxylin was used for counterstaining. After dehydration of gradient ethanol and sealing slices with neutral gum, slices were observed under a microscope. The positive results were stained in yellow. The images were analyzed using Image-Pro Plus software.
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