EVs used in Western blotting analysis were purified from patient plasma using ExoQuick Ultra (System Biosciences, EQULTRA-20A-1), following the manufacturer’s instruction. Final elutes were dissolved in RIPA buffer (1/10 of elute volume). Signals were detected and quantitated with the LI-COR Odyssey system. To calculate marker ratios, intensities of protein bands of each marker were divided by signals of CD9 bands of the same patient. To rescale the marker ratios, the median value of each marker ratio in the whole patient cohort was set to be 100; then all other values were rescaled accordingly. Antibodies used were CD9, Cell Signaling Technology 13174; VGLUT2, Cell Signaling Technology 71555; GPC4, R&D Systems, Bio-Techne, MAB9195; MUC1, BD Biosciences 555925; EGFR, Cell Signaling Technology 4267; EPCAM, BioLegend 118201; CD44v6, Thermo Fisher Scientific BMS125; CD14, BioLegend 367101; annexin A11, Thermo Fisher Scientific MA5-25052; and sLeX, BD Biosciences 551344.
Cd44v6
Manufactured by Thermo Fisher Scientific
Sourced in United States
CD44v6 is a cell surface glycoprotein that functions as a receptor for hyaluronic acid. It is involved in cell-cell interactions, cell adhesion, and migration.
Automatically generated - may contain errors
Lab products found in correlation
Epcam, by BioLegend (3 mentions)
Tspan8, by Miltenyi Biotec (2 mentions)
Iggk2b, by BioLegend (2 mentions)
Cd104, by Miltenyi Biotec (2 mentions)
Iggk1, by Miltenyi Biotec (2 mentions)
C met, by Miltenyi Biotec (2 mentions)
Cd44v6, by Miltenyi Biotec (2 mentions)
Rea isotype control, by Miltenyi Biotec (2 mentions)
Cd104, by Thermo Fisher Scientific (2 mentions)
C met, by Thermo Fisher Scientific (2 mentions)
Epcam, by Miltenyi Biotec (2 mentions)
Exoquick ultra, by System Biosciences (1 mentions)
Vglut2, by Cell Signaling Technology (1 mentions)
Odyssey system, by LI COR (1 mentions)
Cleaved notch1, by Cell Signaling Technology (1 mentions)
Non phospho active β catenin, by Cell Signaling Technology (1 mentions)
Pofut1, by Proteintech (1 mentions)
β catenin, by Cell Signaling Technology (1 mentions)
Lsm 800, by Zeiss (1 mentions)
Gapdh, by Abcam (1 mentions)
7 protocols using cd44v6
1
Extracellular Vesicle Protein Profiling for Biomarker Discovery
2
Multimarker Flow Cytometry Profiling
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The following antibodies were used, adding 1 µL of each antibody to our sample per our previously published work6 (link): EpCAM (Biolegend), CD104 (Thermo Fisher), c-Met (Thermo Fisher), CD44v6 (Thermo Fisher), Tspan8 (Miltenyi). The isotype controls used were the Biolegend IgGk2b (to match EpCAM, CD104) and IgGK1 (c-Met, CD44v6) alongside the Miltenyi REA Isotype control (to match Tspan8). Samples were run at a flow rate of ɸ = 2.5 mL/h.
3
Characterization of Circulating Tumor Cells
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the following antibodies were used, adding 1 μL of each antibody to our sample per our previously published work [6 ]: EpCAM (Biolegend), CD104 (Thermo Fisher), c-Met (Thermo Fisher), CD44v6 (Thermo Fisher), Tspan8 (Miltenyi). The isotype controls used were the Biolegend IgGk2b (to match EpCAM, CD104) and IgGK1 (c-Met, CD44v6) alongside the Miltenyi REA Isotype control (to match Tspan8). Samples were run at a flow rate of = 2.5 mL/hr.
4
Immunofluorescence and Western Blot Analysis
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The protein extraction and Western blot (WB) details can be found in our previous study [6 (link),7 ]. Immunofluorescence (IF) was performed by using Immunol Fluorescence Staining Kit with kFluor594-Labeled Goat Anti-Rabbit IgG (H + L) and kFluor555-Labeled Goat anti-Mouse IgG (H + L) (Keygen Biotech) according to the manual. IF Images were captured using an inverted microscope system (Olympus, IX73) and confocal microscope (Zeiss LSM800). The antibodies used in this article are listed as follows: PLAGL2 (Abcam or ProteinTech), POFUT1 (ProteinTech), GAPDH (Abcam), CD44v6 (Invitrogen), HES5(Abcam), HES1(Abcam), Cleaved Notch1 (Cell Signaling Technology), β-Catenin (Cell Signaling Technology), Non-phospho (Active) β-Catenin (Cell Signaling Technology), MYC (ProteinTech), Cyclin D1 (ProteinTech), Wnt6 (Abcam), OCT4 (Abcm) and Trefoil Factor 3 (Abcam).
5
Evaluating miR-193b-5p and CD44v6 in Breast Cancer
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The miR-193b-5p mimic, pCMV-CD44v6 plasmid and their respective control RNAs were transfected into Hs-578t and BT-549 cells using Lipofectamine 3000 transfection reagent (Invitrogen, USA) according to the manufacturer's protocol. Additionally, 50 nM miRNA and 0.5 μg of pCMV-CD44v6 plasmid were used in a 6-well plate. Protein and RNA were collected 48 h after transfection. RIPA buffer (Beyotime, China) was used for protein extraction. After the total protein concentration was determined by a bicinchoninic acid protein assay kit (Sigma, USA), 30 μg protein samples were separated by 8% SDS polyacrylamide gels and transferred onto PVDF membranes(Millipore, Billerica, USA). The membrane was blocked with 5% nonfat milk in TBST for 1 h and incubated with the indicated antibody (CD44v6, R&D Systems. BBA13, 1:1000; CD44s, CST #5640, 1:1000; GAPDH, 1:5000) at 4 °C overnight. Then HRP-conjugated secondary antibodies (1: 5000) were added. Bands were subsequently visualized using the enhanced plus chemiluminescence assay (Pierce, USA). Measurement of the bands was conducted on an ImageQuant LAS 4000 mini.
6
Western Blot Analysis of Hepatocellular Carcinoma
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c-Met, phospho-c-Met (1349), phospho-c-Met (1234/1235), AKT, phospho-AKT, ERK1/ERK2, phospho-ERK1/ERK2, CD44, E-cadherin, vimetin, and moesin antibodies were purchased from Cell Signaling Technology (Danvers, MA). β-actin antibody was obtained from Sigma (Allentown, PA). CD44v6 was obtained from eBioscience (San Diego, CA) and fibronectin was obtained from BD Sciences (San Jose, CA). All human HCC samples were obtained through an IRB approved protocol (IRB#27146). Tissue samples were incubated with lysis buffer and incubated on ice for 10 min followed by disruption by the TissueLyser (Qiagen, CA) per manufacturer’s recommendation. 15-30 μg of cell lysates were collected and western blot was performed as previously described [30 (link)]. For densitometry analysis, scanned blots were analyzed using Image J (v1.48. NIH, Bethesda, MD) and normalized to Beta-Actin loading control after background subtraction.
7
Immunohistochemical Analysis of Paraffin-Embedded Tissues
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Immunohistochemical staining of formalin-fixed paraffin-embedded tissue was performed in the Biorepository and Tissue Research Facility on a robotic platform (Ventana Discover Ultra Staining Module; Ventana, Tucson, AZ). Tissue sections (4 μm) were deparaffinized using EZ Prep solution (Ventana). A heat-induced antigen retrieval protocol (64 minutes) was performed using Cell Conditioner 1 (Ventana). Endogenous peroxidases were blocked with peroxidase inhibitor (CM1, 8 minutes) before incubation with antibodies for CD44v6 (eBioscience), chromogranin A (Agilent/DAKO), cytokeratin 20 (Agilent/DAKO), Ki67 (Abcam), LGR5 (Miltenyi), or OLFM4 (Cell Signaling) (Table 2 ) for 60 minutes at room temperature. Justification for specific antibodies used is provided in the Supplementary Methods (see Supplementary Digital Content 1, http://links.lww.com/CTG/A612 ). Antigen–antibody complex was detected using DISCOVERY OmniMap anti-mouse multimer for CD44v6 and cytokeratin 20 and DISCOVERY ChromoMap DAB kit (Ventana) with anti-rabbit conjugate for chromogranin A, Ki67, LGR5, and OLFM4. All slides were counterstained with hematoxylin, dehydrated, cleared, and mounted for assessment.
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