Frozen mice colon tissues were fixed in 4% paraformaldehyde for 20 min and permeabilized in 0.5% Triton-X100 for 20 min. The fixed cells were incubated with anti-IL-6, anti-TNF-α (Abcam), EpCAM (Santa Cruz), anti-phospho(276)-p65, anti-phoshp-IκB, anti-EzH2 (Cell Signaling), anti-IL-6 (Abcam) for 16h at 4C followed by secondary anti-mouse Alexa-488 or Cy3 antibody (Invitrogen Life Technology) for 1h at RT. Nuclei were stained with mounting medium with DAPI (VectorLab).
Anti il 6
Manufactured by Abcam
Sourced in United States, United Kingdom, China, Canada
Anti-IL-6 is a laboratory reagent used to detect and quantify the presence of interleukin-6 (IL-6) in biological samples. It is a protein that binds to IL-6, allowing researchers to measure IL-6 levels in various experimental contexts.
Automatically generated - may contain errors
Lab products found in correlation
Anti tnf α, by Abcam (37 mentions)
Anti il 1β, by Abcam (26 mentions)
Pvdf membrane, by Merck Group (7 mentions)
Anti il 10, by Abcam (7 mentions)
Anti il 17, by Abcam (7 mentions)
Anti gapdh, by Abcam (6 mentions)
Anti α sma, by Abcam (5 mentions)
Anti β actin, by Merck Group (4 mentions)
Anti beclin 1, by Cell Signaling Technology (4 mentions)
Anti il 18, by Abcam (4 mentions)
Anti caspase 3, by Abcam (4 mentions)
Anti β actin, by Abcam (4 mentions)
Anti β actin, by Proteintech (4 mentions)
Anti tgf β, by Abcam (4 mentions)
Anti il 8, by Abcam (4 mentions)
Anti collagen 1, by Abcam (4 mentions)
Anti il22, by Abcam (4 mentions)
Anti ifn γ, by Abcam (3 mentions)
Anti stat3, by Cell Signaling Technology (3 mentions)
Anti bcl 2, by Abcam (3 mentions)
107 protocols using anti il 6
1
Immunofluorescence Staining for Colon Tissue
2
Protein Expression Analysis in Colon Tissue
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The colon tissues were homogenized and ultrasonically treated on ice for cell lysis and protein extraction. After determining the protein content, the degeneration process was performed at 100 °C for 5 min. The proteins were separated using 10% SDS-PAGE and transferred to PVDF membranes (0.4-5 μm pore size; Millipore, United States). After blocking with 5% skimmed milk, the membranes were incubated with rabbit anti-TIM3 (1:2000), anti-IL-1β (1:2000), and anti-IL-6 (1:2000) antibodies (Abcam, Cambridge, UK) at 4 °C overnight and then incubated with secondary antibodies labeled with horseradish peroxidase (1:10000, Zhongshan Gold Bridge, Beijing, China) for 2 h at room temperature. The immunoblots were detected using an enhanced chemiluminescent substrate (Millipore) on the ChemiDoc MP system (Bio-Rad, United States). All of the detected protein bands were standardized against β-actin. The semiquantification of each band was performed using ImageJ NIH software (National Institutes of Health, Bethesda, MD, United States).
3
Gastric Biopsy Protein Extraction and Analysis
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The total protein was extracted from samples of gastric biopsies using RIPA buffer containing inhibitors (1 M NaF, Complete Protease Cocktail Inhibitor, and 0.1 M PMSF). The total extracted proteins were quantified using the NanoDrop 2000 spectrophotometer (Thermo Scientific®) using Bradford reagent and standard BSA proteins (5000201, Bio-Rad, USA). The proteins (25 μg) were separated in 12% SDS-PAGE, and transferred to a nitrocellulose membrane, and then blocked with 5% skimmed milk in 1× phosphate buffered saline and 0.1% Tween 20 (PBST), for 2 h at room temperature. Subsequently, the membranes were incubated overnight at 4°C, with primary antibodies: anti-α-tubulin antibody (5168, 1:5,000, Santa Cruz Biotechnology, USA); anti-IL-6 (93356, 1:500, Abcam, USA), and anti-IL-6R (128008, 1:3,000, Abcam). Then, the membranes were incubated individually for 1 h 30 min at room temperature with secondary antibodies: goat anti-rabbit IgG H&L (97051, 1:10,000, Abcam), used for IL-6 and IL-6R, and m-IgGk BP-HRP (1:5.000 Santa Cruz Biotechnology), used for tubulin. Finally, the membranes were washed in PBST, and revealed.
4
Immunohistochemical Analysis of NSCLC Samples
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Paraffin-embedded clinical NSCLC samples and metastatic lung tumors in mouse models were subjected to IHC staining as previously described [21 (link)]. For the detection of FXR, IL-6, IL6ST and p-STAT3, the following primary antibodies were used: anti-bile acid receptor NR1H4 (1:100; cat. no. ab187735; Abcam, Cambridge, UK), anti-IL-6 (1:100; cat. no. ab9324; Abcam), anti-CD130 (gp130) (1:100; cat. no. ab227058; Abcam) and anti-Phospho-STAT3 (Tyr705) (1:200; cat. no. 9145s; Cell Signaling Technology, Inc., Beverly, MA). Concentration-matched non-specific mouse or rabbit IgG served as isotype controls. The IHC staining results were scored independently and blindly by two skilled pathologists, and a final consensus was reached. The staining intensity of tumor cells was scored as negative (0), weak (1), medium (2), and strong (3), respectively. The percentage of positive cells was scored as follows: 0% (0), 1%–25% (1), 26%–50% (2), 51%–75% (3), and 76%–100% (4), respectively. The final IHC scores of human FXR, IL-6, IL6ST and p-STAT3 were obtained by multiplying the staining intensity score with the positive-cell percentage score, and stratified as follows: Low, score < 6 or high, score ≥ 6, in line with our previous studies [18 (link), 19 (link)].
5
Western Blot Analysis of Hepatic Proteins
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The hepatic tissue was homogenized into cold lysis buffer and the lysate was centrifuged and the supernatant was collected in a fresh tube supplemented with protease inhibitor cocktail (Himedia, India) and kept at −80 °C.32 The protein samples were heat denatured and separated by SDS-PAGE followed by electrotransfer to the nitrocellulose membrane. The membranes were blocked in blocking buffer and then exposed to specific primary antibodies diluted in the blocking buffer and kept overnight at 4 °C. The primary antibodies used were rabbit polyclonal anti-phospho AMPK, anti-AMPK, anti-IL-6 and anti-TNF-α (Abcam, USA). The next day membrane was washed and subjected to incubation for 1 h with anti-rabbit IgG secondary antibody conjugated to horseradish peroxidase (Thermo Fisher Scientific, USA) diluted in the same blocking buffer. After a series of washes, they were exposed with the chemiluminescent ECL substrates (Thermo Scientific, USA) and the desired protein bands were detected by chemiluminescent gel documentation system (Bio-Rad, USA). β-Actin was used as a housekeeping protein for equal amounts of protein loaded into the gel. The protein bands were densitometry quantified by Image J, software (NIH, USA) and data presented as arbitrary units.
6
Immunoblotting Antibody Validation Protocol
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Immunoblotting was carried out as described previously [11 (link)]. The following antibodies were purchased from Abcam: anti-GAPDH (1:2500, 37 kDa, AB9485, RRID:AB_307275), anti-IL-6 (1:2000, 17 and 50 kDa, ab9324, RRID:AB_307175), and anti-ICAM-1 (1:4000, 90 kDa, AB53013, RRID:AB_870702). Anti-ACE2 (1:1000, 125 kDa, MA532307, RRID:AB_2809589) and anti-HIF-1α (1:1000, 130 kDa, PA5-85,494, RRID:AB_2792634) antibodies were obtained from Thermo Scientific. The following antibodies were purchased from Cell Signaling Technology: anti-ERK1/2 (1:1000, 42, 44 kDa, #4695, RRID:AB_390779), anti-phospho-ERK1/2 (Thr202/Tyr204), (1:1000, 42, 44 kDa, # 9101, RRID:AB_331646), anti-MSK1 (1:1000, 95 kDa, #3489, RRID:AB_2285349), anti-phospho-MSK1 (Thr581) (1:1000, 95 kDa, #9595, RRID:AB_2181783), anti-p38 (1:1000, 43 kDa, #9212, RRID:AB_330713), anti-phospho-p38 (Thr180/Tyr182) (1:1000, 43 kDa, #4511, RRID:AB_2139682), anti-phospho-JNK (Thr183/Tyr185) (1:1000, 46, 54 kDa, #4668, RRID:AB_823588), anti-JNK (1:1000, 46, 64 kDa, #9252, RRID:AB_2250373), anti-phospho-NFκB p65 (Ser536) (1:1000, 70 kDa, #3033, RRID:AB_331284), and anti-NFκB p65. (1:1000, 70 kDa, #6956, RRID:AB_10828935). The following antibody were obtained from ABclonal (Woburn, MA): anti-phospho-NFkB p65 (S276) (1:1000, 70 kDa, AP0123, RRID:AB_2771505). An anti-phospho-MBP antibody (1:200, 18 kDa, 13–104) was purchased from Merck.
7
Apigenin-Induced Signaling Pathway Analysis
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Cells were seeded at a density of 5 × 105 cells/well in a six-well plate and treated with various concentrations of apigenin (0, 10, 20, and 40 μM) for 24 h. Cells were lysed using protein extraction buffer (Intron Biotechnology, Seoul, South Korea) supplemented with phosphatase inhibitor (genDEPOT, Katy, TX, USA). Proteins extracted from cells were quantified with Coomassie (Bradford) Protein Assay (genDEPOT, Katy, TX, USA), separated by electrophoresis, and transferred onto nitrocellulose membranes (0.45-μM pore size, Merck Millipore, Burlington, MA, USA). The membranes were blotted with first and second antibodies. PI3K (#4255), pAkt (#4060), STAT3 (#4904) (and phopho-STAT3, Ser727 and Tyr701, #9134 and #9167), ERK (#9102) (and phopho-ERK, #4370), snail (#3870), vimentin (#5741), and N-cadherin (#13116) antibodies, and a cell cycle regulation sampler kit (#9932) was obtained from Cell Signaling Technology (CST, Danvers, MA, USA). Anti-β-actin (#A5441, Sigma Aldrich, St. Louis, MO, USA), anti-p53 (#05-224, Millipore, Burlington, MA, USA), and anti-IL-6 (Abcam, Cambridge, UK) were used. The second antibodies (anti-mouse and anti-rabbit) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The blots were visualized with an enhanced chemiluminescent (ECL) detection solution (Intron Biotechnology, Seoul, South Korea)
8
Cytokine Expression in Mouse Stomach
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Stomach tissues obtained from mice were prepared, stained, and imaged to assess cytokine expression. The primary antibodies used for immunofluorescence staining were: lectin GS II, anti-VEGF-β, anti-IL-10 (1:200; Santa Cruz Biotechnology), anti-IFN-γ (1:50; Abcam, Cambridge, UK), anti-IL-6 (1:500; Abcam), anti-TNF-α (1:100; Abcam), anti-IL-1β (1:100; Abcam), and anti-proteinase 3 (Santa Cruz Biotechnology). Expression levels of VEGF-β, GS II, and cytokines were evaluated under confocal microscopy after immunofluorescent staining of deparaffinized tissue sections.
9
Protein Isolation and Western Blot Analysis
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Total protein was isolated from lung tissues and RAW264.7 cells using RIPA lysis buffer (Beyotime, Shanghai, China). The protein concentration was measured using a BCA kit (KeyGen Biotech). An equal volume per sample was subjected to 10% SDS-PAGE and the proteins were transferred onto PVDF membranes (Millipore, Bedford, MA). The membranes were blocked in 5% non-fat milk and probed with anti-GGPPS1 (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA), anti-IL-1β (1:1000, Abcam), anti-IL-6 (1:1000, Abcam), anti-IL-18 (1:200, Abcam), anti-TNF-α (1:1000, Abcam), anti-LC3B (1:200, Abcam), anti-ATG5 (1:1000, Cell Signaling Technology, Danvers, MA), anti-Beclin1 (1:1000, Cell Signaling Technology), anti-Rab37 (1:500, Abcam), and anti-Actin (1:1000, Santa Cruz Biotechnology). After washing, the membranes were probed with HRP-conjugated secondary antibodies (1:2000, Abcam) and the signal was detected by the classical ECL method.
10
Cell Isolation and Characterization Protocol
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