Recombinant human hb egf

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Recombinant human HB-EGF is a growth factor protein produced using recombinant DNA technology. It is a member of the epidermal growth factor (EGF) family and plays a role in cell proliferation and differentiation.

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Recombinant human EGF (229 citations) Human EGF (156 citations) Recombinant EGF (35 citations) HB-EGF (26 citations)

5 protocols using recombinant human hb egf

Investigating HB-EGF Signaling Pathways

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Recombinant human HB-EGF (PeproTech, Rocky Hill, NJ, USA) was dissolved in sterile distilled water. AG1478 (Sigma, St. Louis, MO, USA) were dissolved in 0.1% DMSO. Plumbagin (Sigma, St. Louis, MO, USA) was dissolved in DMSO to 0.1 M and stored at −20°C. Subsequent dilutions were made in cell culture medium. After 24 h culture in six-well plates at a density of 1.5 × 10⁵ cells per well, HSC-T6 cells were separated into seven groups: (A) medium control: 24 h incubation with 10% FBS in DMEM; (B) DMSO control: same as medium control except addition of 0.1% DMSO; (C) HB-EGF (80 ng/mL, dissolved in DMEM): cells were incubated with HB-EGF for 24 h, (D) HB-EGF + AG1478 (5 μM), (E) HB-EGF + PL (2 μM), (F) HB-EGF + PL (4 μM), and (G) HB-EGF + PL (6 μM). The total RNA and cellular proteins were extracted for further analysis.

Chen S., Chen Y., Chen B., Cai Y.J., Zou Z.L., Wang J.G., Lin Z., Wang X.D., Fu L.Y., Hu Y.R., Chen Y.P, & Chen D.Z. (2015). Plumbagin Ameliorates CCl4-Induced Hepatic Fibrosis in Rats via the Epidermal Growth Factor Receptor Signaling Pathway. Evidence-based Complementary and Alternative Medicine : eCAM, 2015, 645727.

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Culturing and Stimulating Human Cardiac Fibroblasts

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Primary human cardiac fibroblasts (HCF) (#C-12375, Promocell) or immortalized human cardiac fibroblasts (iHCF) (#P10453-IM, Innoprot) were used. HCF were cultured in Fibroblast Growth Medium 3 (#C-23130, Promocell) according to the manufacturer’s protocol. HCF were seeded at a density of 5000 cells/cm² in 6-well, 12-well plates and 8-well µ-Slides (#80826, IBIDI) coated with human fibronectin (#F0895, Sigma Aldrich, 0.1% solution; 1:1000 in water) for indirect co-culture, RNA extraction and qRT-PCR analysis. Immortalized iHCF were cultured in Dulbecco modified Eagles medium (DMEM) (#11965092, Thermo Fisher Scientific) supplemented with 10 % FBS (#16000044, Thermo Fisher Scientific) and 50 U/mL Penicillin and Streptomycin according to the manufacturer’s protocol. HCF were seeded at a density of 17 500 cells/cm² in fibronectin-coated (#F0895, Sigma Aldrich, 0.1% solution; 1:1000 in water) 12-well plates and 8-well µ-Slides for indirect co-culture, RNA extraction and qRT-PCR analysis.
For stimulation with recombinant human HB-EGF (#100-47, PeproTech), fibroblasts were seeded in the respective densities one day prior to the stimulation. One day after stimulation, medium was replaced with fresh medium containing 100 ng/ml HB-EGF for 48 h.

Shumliakivska M., Luxán G., Hemmerling I., Scheller M., Li X., Müller-Tidow C., Schuhmacher B., Sun Z., Dendorfer A., Debes A., Glaser S.F., Muhly-Reinholz M., Kirschbaum K., Hoffmann J., Nagel E., Puntmann V.O., Cremer S., Leuschner F., Abplanalp W.T., John D., Zeiher A.M, & Dimmeler S. (2024). DNMT3A clonal hematopoiesis-driver mutations induce cardiac fibrosis by paracrine activation of fibroblasts. Nature Communications, 15, 606.

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Immunological Characterization of PS Signaling

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PS was purchased from Enzyme Research Laboratories (South Bend, IN, USA). Polyclonal antibodies to PS was purchased from Abnova (Taipei, Taiwan). Polyclonal antibodies to LGR in PS (residues 419 to 516) (anti‐ LGR) was purchased from Abnova (Taipei, Taiwan). Factor XII (FXII) was purchased from Enzyme Research Laboratories (South Bend, IN, USA). Polyclonal antibodies to recombinant human EGF (anti‐human EGF) was purchased from Abcam (Tokyo, Japan). Polyclonal antibodies to recombinant human heparin‐binding EGF‐like growth factor (anti‐HB‐EGF) was purchased from PeproTech (Rocky Hill, NJ, USA). Polyclonal antibodies to Recombinant mouse EGF (anti‐mouse EGF) was purchased from R&D Systems, Inc. (Minneapolis, MN, USA). Recombinant mouse EGF was purchased from ProSpec‐Tany TechnoGene Ltd (Ness Ziona, Israel). Recombinant human EGF was purchased from Higeta Shoyu Co., LTD (Ibaraki, Japan). Recombinant human HB‐EGF was purchased from PeproTech.

Sato Y., Sugi T, & Sakai R. (2018). Antigenic binding sites of anti‐protein S autoantibodies in patients with recurrent pregnancy loss. Research and Practice in Thrombosis and Haemostasis, 2(2), 357-365.

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MTT and FITC-Phalloidin Assay with ERK1/2 Inhibition

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MTT (3-(4, 5)-dimethylthiazol (-z-y1)-3, 5-diphenytetrazoliumromide) and fluorescein isothiocyanate (FITC)–phalloidin was purchased from Sigma (St. Louis, MO, USA).
ERK1/2 inhibition was achieved with 10 uM PD98059 (MedChemExpress, Tokyo, Japan). The chemical is also a potent and selective MEK inhibitor with an IC₅₀ of 5 µM by binding to the inactive form of MEK, thereby preventing the activation of MEK1 (IC₅₀ of 2–7 µM) and MEK2 (IC₅₀ of 50 µM) by upstream kinases. When DMSO was used for drug dilution, the final concentration of DMSO did not exceed 0.1% throughout the study. This DMSO concentration was found to not affect cell growth. Recombinant human HB-EGF was obtained from Peprotech (Cranbury, NJ, USA).

Salama Y., Jaradat N., Hattori K, & Heissig B. (2021). Aloysia Citrodora Essential Oil Inhibits Melanoma Cell Growth and Migration by Targeting HB-EGF-EGFR Signaling. International Journal of Molecular Sciences, 22(15), 8151.

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Cell Culture Conditions and Validation

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Cells were cultured at 37°C with 5% CO₂ in humidified atmosphere. All cell lines except for H2107 (NCI-H2107) and 293T were cultured in RPMI 1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific). H2107 cells were cultured in DMEM medium (Thermo Fisher Scientific) supplemented with 5% fetal bovine serum and 1% Glutamax (Thermo Fisher Scientific). 293 T cells were cultured in DMEM medium supplemented with 10% FBS. H1975 cells were cultured in stem cell culture media (Advanced DMEM/F12 [Thermo Fisher Scientific], 1% Glutamax, B-27 supplement [Thermo Fisher Scientific], 10 ng/mL recombinant human HB-EGF [PeproTech, Cranbury, NJ, USA], and 10 ng/mL recombinant human FGF-basic [PeproTech]) (Park et al., 2018 (link)) when indicated. H2107, H82 (NCI-H82), H524 (NCI-H524), H526 (NCI-H526), PC9 (PC-9), H1650 (NCI-H1650), H1975 (NCI-H1975), HCC827, HCC2279, HCC2935, HCC4006, HCC4011, H23 (NCI-H23), H1792, (NCI-H1792) A549, H358 (NCI-H358), H1395 (NCI-H1395), H2347 (NCI-H2347), H460 (NCI-H460), H1155 (NCI-H1155), and 293 T cells were obtained from American Type Tissue Culture (ATCC) or were a kind gift from Dr. Adi Gazdar (UTSW). Mycoplasma contamination check was carried out using a LookOut Mycoplasma PCR Detection Kit (Sigma-Aldrich, St. Louis, MO, USA). Cells were validated by STR profiling.

Inoue Y., Nikolic A., Farnsworth D., Shi R., Johnson F.D., Liu A., Ladanyi M., Somwar R., Gallo M, & Lockwood W.W. (2021). Extracellular signal-regulated kinase mediates chromatin rewiring and lineage transformation in lung cancer. eLife, 10, e66524.

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