ME180 cells (4 × 105 cells/well) were seeded in 12-well dishes 2 days before the assay. Cells were 100% confluent at the time of infection. Cells were infected at an MOI of 10. At 3.5 hpi, PBS or recombinant HB-EGF (R&D Biosystems) reconstituted in PBS was added to infected cultures (final concentration of 6 ng/ml). Cells were incubated for another 30 min. At 4 hpi, adhesion and gentamicin protection assays were performed as described above.
Recombinant hb egf
Manufactured by R&D Systems
Sourced in United States
Recombinant HB-EGF is a protein produced through recombinant DNA technology. It is a member of the epidermal growth factor (EGF) family and plays a role in cellular proliferation and differentiation.
Automatically generated - may contain errors
Lab products found in correlation
Erlotinib, by Chemietek (1 mentions)
Cabozantinib, by Selleck Chemicals (1 mentions)
Brigatinib, by Selleck Chemicals (1 mentions)
Afatinib, by Selleck Chemicals (1 mentions)
Crizotinib, by Selleck Chemicals (1 mentions)
Ceritinib, by Selleck Chemicals (1 mentions)
Hrp conjugated anti rabbit antibody, by Cell Signaling Technology (1 mentions)
Glyceraldehyde 3 phosphate dehydrogenase gapdh, by Cell Signaling Technology (1 mentions)
Fibroblast growth factor (fgf), by R&D Systems (1 mentions)
Gilteritinib, by Cayman Chemical (1 mentions)
Epidermal growth factor (egf), by R&D Systems (1 mentions)
Afatinib, by Calibre Scientific (1 mentions)
Recombinant areg, by R&D Systems (1 mentions)
Wst 1 proliferation assay, by Roche (1 mentions)
Trastuzumab, by Roche (1 mentions)
Cetuximab, by Merck Group (1 mentions)
Bio dot microfiltration apparatus, by Bio-Rad (1 mentions)
0.1 m nitrocellulose membrane, by GE Healthcare (1 mentions)
Anti hb egf antibody, by R&D Systems (1 mentions)
Qualified fbs, by Thermo Fisher Scientific (1 mentions)
5 protocols using recombinant hb egf
1
HB-EGF Modulation of Cellular Adhesion
2
Small Molecule Inhibitor Screening Protocol
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Crizotinib, ceritinib, brigatinib, cabozantinib and afatinib were purchased from Selleck Chemicals (Houston, TX, USA); gefitinib and cetuximab were purchased from Eveleth (Eveleth, MN, USA); erlotinib was purchased from Chemie Tek (Indianapolis, IN, USA); lorlatinib was purchased from Toronto Research Chemicals (Toronto, ON, Canada; and gilteritinib was purchased from Cayman Chemical (Ann Arbor, MI, USA). Recombinant HB‐EGF, EGF, FGF and IGF were purchased from R&D Systems (Minneapolis, MN, USA).
Antibodies against ROS1, phospho‐specific (p) ROS1 (Tyr2274), EGFR, pEGFR (Tyr1068), mitogen‐activated protein kinase (MAPK), pMAPK (Thr202/Tyr204), AKT, pAKT (Ser473), AXL and glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH), and HRP‐conjugated antirabbit antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).
Antibodies against ROS1, phospho‐specific (p) ROS1 (Tyr2274), EGFR, pEGFR (Tyr1068), mitogen‐activated protein kinase (MAPK), pMAPK (Thr202/Tyr204), AKT, pAKT (Ser473), AXL and glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH), and HRP‐conjugated antirabbit antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).
3
Cell Proliferation Assay with Targeted Therapies
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The water-soluble tetrazolium (WST-1) proliferation assay (Roche Diagnostics) was used to measure cell proliferation after knockdown or stimulation as described earlier [17 (link)]. Cells were treated with cetuximab (1/10 µg/ml, Merck), trastuzumab (5/20 µg/ml, Roche), afatinib (0.5 µM, Biozol), DMSO (0.05%, afatinib solvent), trastuzumab solvent (described in [17 (link)]) or cetuximab solvent (8.48 mg/ml NaCl, 1.88 mg/ml Na2HPO4 × 7H2O, 0.41 mg/ml NaH2PO4xH2O) for 72 h (cell numbers see Table S1, Additional file 1 ). In case of stimulation, cells were treated with 5 ng/ml recombinant HBEGF or 15 ng/ml recombinant AREG (R&D Systems).
4
Quantification of Soluble HB-EGF Release
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ME180 cells (1 × 106 cells/well) were seeded in 6-well dishes 2 days before the assay. Cells were 100% confluent on the day of infection. Cells were infected at an MOI of 10 for 2, 4, 6, or 8 h, and total supernatants were collected and stored at −80°C for future analysis. Supernatants were analyzed by dot blot for the presence of soluble HB-EGF using the Bio-Dot microfiltration apparatus (Bio-Rad) fitted with a 0.1-µm nitrocellulose membrane (GE Healthcare Biosciences) rehydrated in TBS. The supernatants were thawed on ice, and 150 µl was added per well for analysis. After the samples were aspirated, the membrane was removed from the apparatus and blocked in TBS containing 5% bovine serum albumin (BSA) for 1 h at room temperature. The membrane was then probed with anti-HB-EGF antibody (R&D Biosystems) in TBS-BSA overnight at 4°C. The secondary antibody (LICOR) was diluted in BSA-TBS and incubated for 1 h at room temperature. The blot was analyzed using densitometry; HB-EGF intensity was normalized to total protein intensity. Recombinant HB-EGF (R&D Biosystems) was serially diluted in PBS to estimate the total HB-EGF concentrations in supernatants by standard curve analysis.
5
Signaling Pathways Regulating HB-EGF
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RPMI 1640 and qualified FBS were from Invitrogen (Carlsbad, CA). Recombinant HB-EGF and betacellulin (BTC) were from R&D Systems (Minneapolis, MN). The HB-EGF inhibitor CRM197 and the Src family kinase inhibitor PP1 were from Sigma-Aldrich (St. Louis, MO). The EGFR tyrosine kinase inhibitor AG1478 and the mTOR complex 1 inhibitor rapamycin were from LC Laboratories (Woburn, MA). Adenoviruses expressing shRNAs against HB-EGF (Adv-shHBEGF) and control scrambled shRNA (Adv-shCTL) were from Vector Biolabs (Malvern, PA). SmartPool siRNA duplexes against rat ChREBP and control siRNA were obtained from Dharmacon (Lafeyette, CO). Primary antibodies and dilutions are listed in Supplementary Table 1 .
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