Brain samples were homogenized and sonicated in lysis buffer. Total protein was measured by a NanoDrop apparatus (MaestroGen, Las Vegas, NV, USA). 20 μg protein were size-fractionated using any-kD Mini-Protean TGX gel electrophoresis and then transferred to a nitrocellulose membrane, and membranes were blocked and washed in TBS-T, and incubated overnight with AC, ACC1, ACC2, PC, PCC, MCC, presynaptic synapsin I, postsynaptic PSD95, PSD93, IL-17, IL-6, TNF-α, NFkB, GFAP, GAP43, ICAM-1, BDNF, CXCL 16, OPG and MMP-9 antibodies (Abcam, Cambridge, UK). The following day, membranes were washed and then incubated with horseradish peroxidase-conjugated goat anti-rabbit (Abcam, Cambridge, UK) or goat anti-mouse (Abcam, Cambridge, UK) secondary antibody (diluted 1:1000) for 1 h at room temperature. Protein loading was controlled with an anti-β-actin antibody (Abcam, Cambridge, UK). Protein levels were analyzed densitometrically using an image analysis system (Image J; National Institute of Health, Bethesda, MD, USA), corrected with values determined on β-actin blots, and expressed as relative values compared with the control group.
Cxcl16
Manufactured by Abcam
Sourced in United Kingdom, United States
CXCL16 is a chemokine that plays a role in the recruitment and activation of immune cells. It is involved in various biological processes, including inflammation, immune response, and cell migration. This product is a recombinant protein that can be used for research purposes.
Automatically generated - may contain errors
Lab products found in correlation
Brain derived neurotrophic factor (bdnf), by Abcam (2 mentions)
Il 17, by Abcam (2 mentions)
Glial fibrillary acidic protein (gfap), by Abcam (2 mentions)
Icam 1, by Abcam (2 mentions)
Interleukin 6 (il 6), by Abcam (2 mentions)
Anti β actin antibody, by Abcam (2 mentions)
Gap43, by Abcam (2 mentions)
Tnf α, by Abcam (2 mentions)
Goat anti mouse, by Abcam (1 mentions)
Nf kb, by Abcam (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit, by Abcam (1 mentions)
Mmp 9, by Abcam (1 mentions)
Ag490, by Selleck Chemicals (1 mentions)
Vimentin, by Roche (1 mentions)
Cytokeratin 7, by Agilent Technologies (1 mentions)
Pt link module, by Agilent Technologies (1 mentions)
Cd163, by Roche (1 mentions)
Autostainer plus, by Agilent Technologies (1 mentions)
Tunel assay, by Merck Group (1 mentions)
F4 80, by Thermo Fisher Scientific (1 mentions)
9 protocols using cxcl16
1
Western Blot Analysis of Brain Proteins
2
CXCL16 and STAT3 Inhibitor Effects
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This research was in agreement with the Declaration of Helsinki. The cell line used in this study was mouse L929, which was purchased from the cell bank of the Shanghai Biology Institute (Shanghai, China). Cells were cultured in DMEM medium (Trueline, USA) and grown in a 5% CO2 condition at 37°C. Then, cells were cultured by the mouse recombinant protein CXCL16 (Abcam, UK) and the STAT3 inhibitor AG490 (Selleck, USA) for 48 h with different concentrations, including 50, 100, and 200 ng/ml, respectively.
3
Immunohistochemical Profiling of Formalin-Fixed Paraffin-Embedded Tissue Sections
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Tumor tissue or cultured cells were fixed in formalin and paraffin embedded according to routine protocol. Deparaffinization and rehydration of tissue sections of 4 μm thickness was performed according to standard procedures. Sections were subjected to epitope retrieval in a PT Link module (Dako, Santa Clara, CA, USA) and immunohistochemical stainings were performed using an Autostainer Plus (Dako) according to the manufacturer’s standard protocol. Antibodies used were: cytokeratin 7 (Dako, M7018, 1:100) cytokeratin 19 (Ventana, Basel, Switzerland, 760-4281, prediluted), vimentin (Ventana, 790-2917, prediluted), CD10 (Ventana, 790-4506, prediluted) CD31 (Ventana, 760-4378, prediluted), CD68 (Dako, M0814, 1:1000), CD163 (Ventana, 760-4437, prediluted), and CXCL16 (Abcam, Cambridge, UK, ab101404, 1:100). Sections were stained with hematoxylin and eosin or counterstained with hematoxylin.
4
Immunohistochemical Analysis of CXCL16, ERG, F4/80, and Ki-67 in Xenograft Tumors
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Formalin-fixed, paraffin-embedded tissue sections from tissue microarrays or xenograft tumors were stained using immunohistochemistry for CXCL16 (Abcam, Cambridge, MA, USA; dilution ratio 1:100), ERG (Ventana Medical Systems, Tucson, AZ, USA), F4/80 (eBioscience, San Diego, CA, USA, dilution ratio 1:200), and Ki-67 (Neomarkers, Fremont CA, USA, dilution ratio 1:500), using the BenchMark XT automated immunohistochemistry slide staining system (Ventana) according to the manufacturer’s instructions. TUNEL assay (Millipore, MA, USA) was used to detect the apoptotic cells in xenograft tumor sections. To analyze the immunoreactivities, the core tumor areas were divided into quarters, and 5 areas were randomly chosen from each quarter and the central area. Under 400x magnification, immunoreactive cells were counted by two different medical doctors including one pathologist and expressed as the percentage of positive cells per area.
5
Western Blot Analysis of Piezo1 and Related Proteins
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Western blot was performed as in our previous work [18 (link)] with a little modification. Briefly, 100 μg protein extracted from HCC cells was loaded and resolved on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) for Piezo1 detection, and 20 μg protein extract for other target protein detection. The conditions of membrane transfer were optimized as 350 mA, 210 min for Piezo1, 300 mA, 45 min for von Hippel‐Lindau (VHL), 300 mA, and 90 min for other target proteins. The diluted primary antibodies were as follows: integrin β1 (1:1000, Cell Signal Technology, Boston, MA, USA), Piezo1 (1:500, Abcam), COL1 (1:1000, Affinity), α‐tubulin (1:5000, Proteintech), HIF‐1α (1:1000, Abcam), VHL (1:1000, Abcam), IGFBP2 (1:1000, Abcam), CXCL16 (1:1000, Abcam), L‐vascular endothelial growth factor A (L‐VEGFA) (1:1000, Proteintech), ubiquitin (UB) (1:1000, Cell Signal Technology). The secondary horseradish peroxidase (HRP)‐conjugated antibody (Proteintech) was diluted at 1:5000.
6
Immunohistochemical Analysis of Patient-Derived Aggregates
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Patient-derived aggregates and tissue sections were paraffin embedded, and de-paraffinized in Xylene. Anti-Cytokeratin (mouse monoconal), and Vimentin (mouse monoclonal clone V9) antibodies were purchased from Dako (Carpinteria, CA, USA), anti-Ki67 (rabbit monoclonal) antibody was purchased from Biocare (Concord, CA, USA). The fluorophores Cy3, FITC, and Alexa 647 conjugates were used at a concentration of 1 μg/mL (Biocare). Imaging was performed through the Zeiss Axio Observer. The chemokine composition of the patient-derived aggregate was identified by using primary antibodies on the tissue or aggregate of interest for 1 h: Vimentin (mouse monoclonal clone V9), SDF-1 (rabbit monoclonal; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and the CXCL16 (rabbit monoclonal; abcam, Cambridge, MA, USA).
7
Proteomic Analysis of Pancreatic Cancer Cells
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RIPA buffer (R0010, Beijing Solarbio Science & Technology, China) supplemented with protease and phosphatase inhibitors was used to isolate total proteins from treated or untreated pancreatic cancer cells. The proteins were quantified using the bicinchoninic acid kit, separated using 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto NC-transfer membrane (HATF00010, Millipore, USA). The NC membranes were blocked with 1% bull serum albumin and incubated with specific primary antibodies at 4 °C overnight followed by the secondary antibody incubation at room temperature for 1 hr. Primary antibodies against N-cadherin, E-cadherin, β-catenin, Snail, Slug, ZO-1, Akt, phospho-Akt, p38, Phospho-p38, Erk, Phospho-Erk were purchased from Cell Signaling Technology. Primary antibodies against CXCL16, CXCR6, and ADAM10 were purchased from Abcam. Mouse anti-GAPDH was purchased from ProteinTech. All primary antibodies were diluted at 1:1000, and the corresponding secondary antibodies were diluted at 1:5000. All antibodies are listed in Supplementary Table S2 .
8
Western Blot Analysis of Brain Tissue
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Brain tissue harvested from hippocampi was analyzed by the Western blot method [39] (link). Brie y, tissue samples of each group were pooled and lysed in a buffer containing enzyme inhibitors.
Total protein content was assessed using the Qubit 2.0 Fluorometer (Invitrogen, CA, USA). Twenty µg samples were electrophoresed and then transferred to a nitrocellulose membrane using the Trans-Blot Turbo Transfer System. After that, membranes were blocked for one h at room temperature, washed, and incubated overnight with AC, ACC1, ACC2, PC, PCC, presynaptic synapsin I, postsynaptic PSD95, PSD93, IL-17, IL-6, TNF-a, GFAP, GAP43, ICAM-1, BDNF and CXCL 16 (Abcam, Cambridge, UK). Then, membranes were washed with TBS-T, incubated with a rabbit secondary (Abcam, Cambridge, UK) antibody for 1 h at room temperature. Individual blots were performed at least three times. Protein loading was controlled by stripping and reprobing the nitrocellulose membrane with an anti-β-actin antibody (Abcam, Cambridge, UK). Protein levels were analyzed densitometrically using an image analysis system (Image J; National Institute of Health, Bethesda, MD, USA), corrected with values determined on β-actin blots, and expressed as relative values compared with the control group.
Total protein content was assessed using the Qubit 2.0 Fluorometer (Invitrogen, CA, USA). Twenty µg samples were electrophoresed and then transferred to a nitrocellulose membrane using the Trans-Blot Turbo Transfer System. After that, membranes were blocked for one h at room temperature, washed, and incubated overnight with AC, ACC1, ACC2, PC, PCC, presynaptic synapsin I, postsynaptic PSD95, PSD93, IL-17, IL-6, TNF-a, GFAP, GAP43, ICAM-1, BDNF and CXCL 16 (Abcam, Cambridge, UK). Then, membranes were washed with TBS-T, incubated with a rabbit secondary (Abcam, Cambridge, UK) antibody for 1 h at room temperature. Individual blots were performed at least three times. Protein loading was controlled by stripping and reprobing the nitrocellulose membrane with an anti-β-actin antibody (Abcam, Cambridge, UK). Protein levels were analyzed densitometrically using an image analysis system (Image J; National Institute of Health, Bethesda, MD, USA), corrected with values determined on β-actin blots, and expressed as relative values compared with the control group.
9
Quantifying Tumor Immune Markers
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Immunohistochemical analysis for CD163 (Cell Signaling; diluted 1:1000), CXCR6 (R&D system; diluted 1:250), and CXCL16 (Abcam; diluted 1:1000) were done on formalinfixed, paraffin-embedded tissue sections with the Bench-Mark XT automated immunohistochemistry slide staining system (Ventana; Tucson, AZ, USA), according to the manufacturer's instructions. To analyze immunoreactivities, the core areas of tumors were divided into quarters, and five areas were randomly chosen from each quarter and central area. Under 400! magnification, CD163 C or CXCR6 C cells were counted by two different medical doctors including one pathologist and expressed as the number of cells per 10 3 mm 2 section area. The immunohistostaining intensity of CXCL16 was scored from 0 to 3, at a 400! magnification, and the percentage of positive cells were calculated as: 0, !5%; 1, 5-25%; 2, 25-50%; 3, 50-70%; 4, more than 75%, at a 100! magnification. The final score, 0-3, was defined as the sum of the intensity and percentage.
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