GCIY was co-cultured with TDMs (ratio of TDMs/GCIY: 0, 2, 5, 10) or cultured in medium containing recombinant human IL-6 at a range of 0–100 ng/ml for 48 hours. Cells were washed with cold PBS and lysed with the SDS buffer containing protease inhibitors (cOmplete Mini, Roche Diagnostics GmbH, #11836153001). Equivalent amounts of protein from whole-cell lysates were loaded into each lane of 10% SDS–polyacrylamide gel and electrophoretically transferred to Hybond-polyvinylidene difluoride transfer membranes (GE Healthcare UK Ltd.). Membranes were incubated with the following primary antibodies overnight at 4°C: anti-signal transducer and activator of transcription 3 (STAT3; rabbit monoclonal IgG, diluted 1:1000; Cell Signaling Technology, # 14801, RRID:AB_2798618) and anti-phosphorylated STAT 3 (p-STAT3; rabbit monoclonal IgG, diluted 1:2000; Cell Signaling Technology, #9145, RRID:AB_2491009). Equal loading of samples was confirmed by probing with anti–β-actin antibody (Sigma-Aldrich, #A5441). Membranes were subsequently incubated with secondary antibodies for 1 hour. Peroxidase activity of secondary antibodies was detected using an ECL prime Western Blotting Detection Reagent (GE Healthcare UK Ltd., #RPN2232) and visualized using an Amersham Imager 600 (GE Healthcare UK Ltd.) according to the manufacturer’s protocol.
Hybond polyvinylidene difluoride transfer membranes
Manufactured by GE Healthcare
Sourced in United Kingdom
Hybond-polyvinylidene difluoride transfer membranes are a type of laboratory equipment used for protein and nucleic acid transfer and immobilization. These membranes are designed to provide a reliable and efficient platform for various blotting techniques, such as Western blotting, Northern blotting, and Southern blotting.
Automatically generated - may contain errors
Lab products found in correlation
Hybond, by Cytiva (5 mentions)
Complete mini, by Roche (2 mentions)
Ecl prime western blotting detection reagent, by GE Healthcare (2 mentions)
Anti β actin antibody, by Merck Group (2 mentions)
β actin, by Merck Group (2 mentions)
Amersham ecl chemiluminescence system, by GE Healthcare (2 mentions)
Amersham ecl, by Cytiva (2 mentions)
Amersham imager 600, by GE Healthcare (1 mentions)
Rabbit monoclonal igg, by Cell Signaling Technology (1 mentions)
Amersham imager, by Cytiva (1 mentions)
Las 4000 mini, by Fujifilm (1 mentions)
Las 4000, by Cytiva (1 mentions)
α sma, by Abcam (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
Peroxidase linked secondary antibodies, by Cytiva (1 mentions)
N cadherin, by Cell Signaling Technology (1 mentions)
Vimentin, by Cell Signaling Technology (1 mentions)
5 protocols using hybond polyvinylidene difluoride transfer membranes
1
STAT3 Activation in GCIY Cells by IL-6 and TDMs
2
Adenovirus E1A Protein Detection
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Proteins extracted from whole-cell lysates were electrophoresed on 10 % SDS–polyacrylamide gels and were transferred to Hybond-polyvinylidene difluoride transfer membranes (GE Healthcare UK Ltd.). The membranes were incubated with primary antibodies against adenovirus type 5 E1A (BD Biosciences, Franklin Lakes, NJ) and β-actin (Sigma-Aldrich), followed by peroxidase-linked secondary antibody. The Amersham ECL chemiluminescence system (GE Healthcare UK Ltd.) was used to detect the peroxidase activity of the bound antibody. Equal loading of samples was confirmed by β-actin analysis.
3
EGFR and HER2 Expression in Esophageal Cancer
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The expression of EGFR and HER2 in esophageal cancer cells was examined using WB. These cells were lysed using cell lysis buffer (50 mmol/L Tris–HCl (pH 7.4), 30 mmol/L NaCl, and 1% Triton X-100) containing protease inhibitors (cOmplete Mini, Roche Diagnostics GmbH, Basel, Switzerland), and whole proteins extracted by centrifugation (14.0 × 103 rpm same as 15.0 × 103 G) for 10 min at 4 °C. The concentrations of extracted protein were measured using standard protocols. Samples containing 40 µg protein were electrophoresed on polyacrylamide gels and transferred to Hybond-polyvinylidene difluoride transfer membranes (GE Healthcare, UK). The membrane was incubated overnight at 4 °C with primary antibodies against EGFR (1:1000 dilution), HER2 (1:1000 dilution), and β-actin (1:1000 dilution). Membranes were washed in Tris Buffered Saline with Tween (TBST) and were incubated with secondary antibodies for 1 h at room temperature (RT) (20–22 °C). After washing, the membranes were visualized using an LAS-4000 mini (FUJIFILM, Tokyo, Japan).
4
HER2-ECD Expression in Gastric Cancer Cells
5
Epithelial-Mesenchymal Transition in Tumor Cells
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Tumor cells were stimulated for 4 days with CM/CAFTE4 or CM/CAFOE19 (TE4CM/CAF or OE19CM/CAF) or cocultured with fibroblasts (TE4DC or OE19DC). In the coculture model, the tumor cells mixed with FEF3 cells were isolated using the same method as that used for flow cytometry. FEF3 cells were stimulated with CM from tumor cells (CM/TE4 or CM/OE19) for 2 days (CAFTE4 or CAFOE19). Primary antibodies against E-cadherin (Cell Signaling Technology), N-cadherin (Cell Signaling Technology), Vimentin (Cell Signaling Technology), αSMA (Abcam), and β-actin (Sigma) were used. Cells were washed, lysed in SDS buffer, and centrifuged. The supernatants were collected and subjected to WB. Proteins were electrophoretically transferred to Hybond-polyvinylidene difluoride transfer membranes (GE Healthcare Life Science) and incubated with a primary antibody, followed by peroxidase-linked secondary antibodies (Amersham Bioscience). An ECL Prime Western Blotting Detection Reagent (GE Healthcare UK Ltd.) was used to detect the peroxidase activity of the bound antibody.
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