The protein was extracted using RIPA buffer (Beyotime), separated by 10% SDS-PAGE, and then transferred to PVDF membranes. The membrane was cultured with primary antibodies against HOXC10, Interleukin-6 (IL-6, ab233706, Abcam), Janus kinase 2 (JAK2, ab108596, Abcam), phospho-JAK2 (p-JAK2, ab195055, Abcam), Signal transducer and activator of transcription 3 (STAT3, ab68153, Abcam), phospho-STAT3 (p-STAT3, ab267373, Abcam), and GAPDH (ab8245, Abcam) at 4°C overnight, followed by incubation with secondary antibody for 1 h at 37°C. The enhanced chemiluminescent (ECL) reagent (Beyotime) was employed to visualize protein bands, and quantification was performed using IMAGE J software (National Institutes of Health) [13 ].
Ab233706
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab233706 is a laboratory reagent that can be used for research purposes. It is a manufactured product designed for use in various scientific experiments and analyses.
Automatically generated - may contain errors
Lab products found in correlation
Ab8245, by Abcam (7 mentions)
Ab183218, by Abcam (6 mentions)
Ab215188, by Abcam (5 mentions)
Ab178945, by Abcam (5 mentions)
Ab254360, by Abcam (5 mentions)
Ab216995, by Abcam (4 mentions)
Ab16502, by Abcam (4 mentions)
Ab7817, by Abcam (3 mentions)
Ab22048, by Abcam (3 mentions)
Pierce ecl, by Thermo Fisher Scientific (3 mentions)
Ab267373, by Abcam (2 mentions)
Ab109201, by Abcam (2 mentions)
Ab263899, by Abcam (2 mentions)
Ab133462, by Abcam (2 mentions)
Ab38449, by Abcam (2 mentions)
Ab234437, by Abcam (2 mentions)
Ab133739, by Abcam (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Ab32536, by Abcam (2 mentions)
Ab32518, by Abcam (2 mentions)
30 protocols using ab233706
1
Analyzing HOXC10 and JAK/STAT Signaling
2
Hepatic Stellate Cell Polarization Assay
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Human hepatic stellate cell line LX‐2 was cultured in DMEM complete medium at 37 °C under 95% air and 5% CO2. For hepatic stellate cell polarization in vitro, LX‐2 cells were treated with CRC‐derived CM, rhFGF19 proteins (50 ng mL−1, R&D Systems) or rhIL‐1α (1 ng mL−1, R&D Systems) for 24 h. Human FGF19 neutralizing antibody (10 µg mL−1, AF969, R&D Systems), FGFR4 inhibitor fisogatinib (100 nм, S8503, Selleck) and BLU9931 (10 µм, A8706, APExBIO), JAK2 inhibitor fedratinib (10 µм, S2736, Selleck), STAT3 inhibitor C188‐9 (5 µg mL−1, S8605, Selleck), and anakinra (20 mg mL−1, Kineret) were used to inhibit iCAFs formation. WB, qRT‐PCR and ELISA were performed to detect the expression of myCAFs markers (α‐SMA, ACTG2, COL1A1, and COL2A1) and iCAFs markers (IL1A, IL1B, IL6, CXCL1, CXCL5), and the phosphorylation of JAK2‐STAT3 signaling pathway. Flow cytometry (FC) was performed to detect the expression of iCAF surface marker PDGFRα (1:250, 3174, Cell Signaling Technology) and myCAF marker α‐SMA (1:200, ab7817, Abcam). Data was collected and analyzed on a FACSCalibur flow cytometer (BD) FlowJo software (USA). Immunofluorescence (IF) staining was performed to detect the expression of pan‐CAF marker podoplanin (PDPN; 1:500, ab10288, Abcam) and iCAF marker IL‐6 (1:100, ab233706, Abcam). Images were acquired on a fluorescence microscope (Olympus).
3
Protein Expression Analysis in Soft Palate
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Total proteins from soft palate tissues were extracted with a Total Protein Extraction Kit (Merck Millipore, Burlington, MA, USA) in accordance with the manufacturer's instructions . Proteins were loaded onto 10% SDS/PAGE and then transferred to poly(vinylidene difluoride) membranes (Merck Millipore). The transferred membranes were blocked in 5% BSA for 1 h at room temperature. Membranes were incubated at 4 °C overnight with primary antibodies against HMGB1 (dilution 1 : 2000; ab18256; Abcam), CD68 (dilution 1 : 1000; ab125212; Abcam), CD31, TLR4 (dilution 1 : 1000; GB11519; Servicebio), p‐NF‐κB p65 (dilution 1 : 2000; 8214S; Cell Signaling Technology, Danvers, MA, USA), VEGF (dilution 1 : 2000; 500661S; Cell Signaling Technology), MMP9 (dilution 1 : 2000; 13667S; Cell Signaling Technology), IL‐6 (dilution 1 : 2000; ab233706; Abcam) and GAPDH (dilution 1 : 10 000; 5174S; Cell Signaling Technology). Appropriate horseradish peroxidase‐conjugated secondary antibodies were incubated at 37 °C for 30 min. Finally, protein bands were detected with a chemiluminescent substrate.
4
Quantification of Inflammatory Cytokines in HFLS Cells
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HFLS cells were lysed with RIPA lysate (Beyotime). Total proteins were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Millipore). The membranes were incubated with 5% skimmed milk for 1 h, and incubated overnight at 4℃ with primary antibodies against interleukin‐1β (IL‐1β) (1:1000, ab216995; Abcam), interleukin‐6 (IL‐6) (1:1000, ab233706; Abcam), tumour necrosis factor‐α (TNF‐α) (1:1000, ab215188; Abcam), and GAPDH (1:1000, ab8245; Abcam). After washing, the membranes were incubated with a secondary antibody (1:5,000; Biotech) at room temperature for 4 h. The signals were developed using Pierce ECL Western Blot Substrate (Thermo Fisher Scientific), imaged, and analyzed with GAPDH as the control using ImageJ software.
5
Western Blotting Analysis of Inflammatory Markers
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Western blotting was performed using antibodies against calpain-2 (Cat. 2,539, 1:1,000; Cell Signaling Technology), IL-6 (ab233706,1:1,000; Abcam, Cambridge, United Kingdom), TNF-α (ab183218,1:1,000; Abcam, Cambridge, United Kingdom), IL-1β (ab216995,1:1,000; Abcam, Cambridge, United Kingdom), p-STAT3 (Tyr705) (Cat. 9,145, 1:1,000; Cell Signaling Technology), STAT3 (Cat. 5,345, 1:1,000; Cell Signaling Technology), and GAPDH (Cat. 2,118, 1:5,000; Cell Signaling Technology). Next, the proteins were probed with horseradish peroxidase (HRP)-conjugated secondary antibodies and visualised using a ChemiDoc Imaging System (Bio-Rad Laboratories, Hercules, CA, United States). The relative quantity of proteins is presented as the ratio of target proteins to GAPDH.
6
Exosome Protein Profiling via Western Blot
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Total proteins from the exosomes or cells were extracted and detected using the bicinchoninic acid kit. Briefly, 40 µg of total protein was separated using sodium dodecyl-sulfate polyacrylamide gel electrophoresis (120 V, 90 min) and transferred to polyvinylidene fluoride (PVDF) membranes (90 V, 90 min). For blocking, 5% non-fat milk was added to the PVDF membranes for 1 h. Anti-CD9 (1: 1,000, ab307085, Abcam, Cambridge, MA, USA), anti-CD63 (1: 1,000, ab217345, ab68418), anti-CD81 (1: 1,000, ab109201), anti-SIRT3 (1: 500, ab189860), anti-AMPK (1: 1,000, ab207442), anti-p-AMPK (1: 1,000, ab133448), anti-LC3II/I (1: 1,000, ab48394), anti-P62 (1: 1,000, ab240635), anti-NLRP3 (1: 1,000, ab263899), anti-apoptosis-associated speck-like protein containing a CARD domain (ASC) (1: 1,000, ab70627), anti-IL-1β (1: 1,000, ab283822), anti-iNOS (1:1,000, ab178945), anti-IL-6 (1: 1,000, ab233706), anti-TNF-α (1:1,000, ab183218), anti-Calnexin (1:1000, ab22595) and anti-GAPDH (1:1,000, ab8245) were added and incubated overnight at 4 °C. Moreover, the horseradish peroxidase-labeled goat-anti-rabbit secondary antibody (1:5,000 diluted) was incubated at 25 ℃ for 2 h. Protein blots were detected using Pierce™ ECL (Thermo Fisher, Waltham, USA) in ChemiDoc MP (Bio-Rad, California, USA).
7
Western Blotting of Inflammatory Markers
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The cellular protein lysates from SH‐SY5Y cell samples were acquired by means of RIPA lysis buffer. Later, the extracted proteins were treated with 15% SDS‐PAGE gel, and then shifted electrophoretically to PVDF membranes. Next, the obtained membranes were blocked with 5% skim milk. Primary antibodies against loading control GAPDH (ab8245, Abcam, Cambridge, MA) and IL‐6 (ab233706, Abcam), IL‐1β (ab254360, Abcam), TNF‐α (ab183218, Abcam), along with the corresponding secondary antibodies (ab7090, Abcam) were utilized after dilution. Western band was analyzed with enhanced chemiluminescence Western Blotting Detection Kit (Solarbio).
8
Western Blot Analysis of NF-κB Pathway
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The FLS were
collected and boiled in sample-loading buffer for 10 min at 95 °C.
The proteins were electrophoretically resolved on a 12% SDS-PAGE gel
at 120 V and transferred to PVDF membranes. The PVDF membrane was
then blotted with primary antibodies at 4 °C overnight. The PVDF
membrane was washed with TBST and then incubated with peroxidase-conjugated
secondary antibodies. The chemiluminescent signal was visualized according
to the manufacturer’s instruction. Primary antibodies targeting
the following proteins were used: p-IKBα (phosphor S36, ab133462,
Abcam), IKBα (ab76429, Abcam), p-p65 (phospho S276, ab183559,
Abcam), p65 (ab16502, Abcam), IL-6 (ab233706, Abcam), MMP-1 (ab134184,
Abcam), and β-actin (ab115777, Abcam).
collected and boiled in sample-loading buffer for 10 min at 95 °C.
The proteins were electrophoretically resolved on a 12% SDS-PAGE gel
at 120 V and transferred to PVDF membranes. The PVDF membrane was
then blotted with primary antibodies at 4 °C overnight. The PVDF
membrane was washed with TBST and then incubated with peroxidase-conjugated
secondary antibodies. The chemiluminescent signal was visualized according
to the manufacturer’s instruction. Primary antibodies targeting
the following proteins were used: p-IKBα (phosphor S36, ab133462,
Abcam), IKBα (ab76429, Abcam), p-p65 (phospho S276, ab183559,
Abcam), p65 (ab16502, Abcam), IL-6 (ab233706, Abcam), MMP-1 (ab134184,
Abcam), and β-actin (ab115777, Abcam).
9
Western Blot Analysis of Signaling Pathways
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The cells and tissues were lysed with protein was extracted. BCA kit was used to determine the protein concentration. Same amount protein samples were separated with 12% sodium dodecyl‐sulfate polyacrylamide gel electrophoresis. The protein samples were transferred to nitrocellulose membrane (Millipore) for 30–50 min. Primary antibodies were used to culture membranes overnight at 4°C. After washing with phosphate‐buffered saline (PBS) for three times, the membranes were incubated with second antibodies for 2 h at room temperature. After washing with PBS for three times, electrochemiluminescence exposure solution was added to the membranes. Quantity one software was used to analyze band gray. The antibodies used in this study were listed as follows: anti‐IL‐6 antibody (ab233706, Abcam), anti‐pAKT antibody (ab38449, Abcam), anti‐gp130 antibody (ab227058, Abcam), anti‐pJAK2 antibody (ab32101, Abcam), anti‐pSTAT3 antibody (ab267373, Abcam), anti‐GAPDH antibody (ab9485, Abcam), and goat anti‐Rabbit IgG (ab205718, Abcam).
10
Comprehensive Western Blot Antibody Panel
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Western blot was performed as described previously [28 (link)]. The primary antibodies were as follows: anti-IGFBP1 (ab180948, Abcam), anti-Rev-erbα (sc-393215, Santa Cruze), anti-β-Actin (ab179467, Abcam), anti-β-Tubulin (ab179513, Abcam), anti-PR (human, 8757, Cell Signaling Technology), anti-C/EBPβ (ab32358, Abcam); anti-IL-6 (human, ab233706, Abcam), anti-IL-6R (human, ab222101, Abcam), anti-PR (mouse, ab133526, Abcam), anti-IL-6 (mouse, ab229381, Abcam), anti-IL-6R (mouse, ab300581, Abcam), anti-Wnt4 (sc-376279, Santa Cruze). β-Tubulin and β-Actin were used as internal standards.
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