Afatinib

Manufactured by Bio-Techne

Sourced in United Kingdom

Afatinib is a lab equipment product manufactured by Bio-Techne. It is a small-molecule tyrosine kinase inhibitor that targets the ErbB family of receptor tyrosine kinases, including EGFR, HER2, and HER4.

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Lab products found in correlation

Gefitinib, by Bio-Techne (3 mentions) Wortmannin, by Bio-Techne (2 mentions) 485 mi 100 cf, by R&D Systems (1 mentions) Pemigatinib, by Selleck Chemicals (1 mentions) 415 ml 005 cf, by R&D Systems (1 mentions) 406 ml 005 cf, by R&D Systems (1 mentions) Poly l lysine (pll), by Provitro (1 mentions) Unc2025, by Selleck Chemicals (1 mentions) Astrocyte medium, by ScienCell (1 mentions) Hek293t, by American Type Culture Collection (1 mentions) Skbr3, by American Type Culture Collection (1 mentions) Bt474, by American Type Culture Collection (1 mentions) Bt549, by American Type Culture Collection (1 mentions) Mda mb 231, by American Type Culture Collection (1 mentions) Hcc827, by American Type Culture Collection (1 mentions) Bms 911543, by Selleck Chemicals (1 mentions) Ehmuc1, by Thermo Fisher Scientific (1 mentions) Lapatinib, by LC Laboratories (1 mentions) Erlotinib, by LC Laboratories (1 mentions) Gefitinib, by Axon Medchem (1 mentions)

5 protocols using afatinib

Compound Library Preparation and Cell Lines

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The ReFRAME library was received from Calibr, Scripps Research, as compounds dissolved to 10 mM in DMSO, spotted in 30 nL volumes in black 384 well plates. All peptides used were prepared with C-terminal amides from Cambridge Research Biochemicals (Billingham, UK) and provided at > 95% purity. Cambridge Research Biochemicals (Billingham, UK) synthesized the ester and acid forms of SDZ-224015 (compounds 4 & 5) used in follow-up studies, provided at > 95% purity. Additional compound 4 was synthesised as described below. pelitinib (10), afatinib (11), dacomitinib (12) and gefitinib (13) were obtained from Tocris (Bristol, UK). The active form of Tarloxotinib (9) was from Molport (Riga, Latvia). Compound 3 was from Santa Cruz Biotechnology (Dallas, Texas, USA), Compounds 2, 7 and 8 were from MedChem Express (Sweden); Compound 6 was from Bachem (Bubendorf, Switzerland).
The African monkey kidney cell line Vero E6-GFP was a gift kindly provided by M. van Loock, Janssen Pharmaceutica, Beerse, Belgium.
The hepatocellular carcinoma cell line Huh7 was a gift kindly provided by Ralf Bartenschlager, University of Heidelberg, Germany.
All compounds were obtained at a manufacturer specification of > 98% purity.
Unless otherwise stated all other reagents were from Sigma Aldrich (Poole, UK).

Redhead M.A., Owen C.D., Brewitz L., Collette A.H., Lukacik P., Strain-Damerell C., Robinson S.W., Collins P.M., Schäfer P., Swindells M., Radoux C.J., Hopkins I.N., Fearon D., Douangamath A., von Delft F., Malla T.R., Vangeel L., Vercruysse T., Thibaut J., Leyssen P., Nguyen T.T., Hull M., Tumber A., Hallett D.J., Schofield C.J., Stuart D.I., Hopkins A.L, & Walsh M.A. (2021). Bispecific repurposed medicines targeting the viral and immunological arms of COVID-19. Scientific Reports, 11, 13208.

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Astrocyte Stimulation and Modulation

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Primary human astrocytes were a gift of B. Winner, Department of Stem Cell Biology, University Hospital Erlangen, Erlangen, Germany, and originally were obtained from ScienCell (1800) and cultured according to the manufacturer’s instructions. In brief, cells were passaged in astrocyte medium (ScienCell, 1801) until confluency and subsequently plated onto plates precoated with 2 μg/mL poly-l-lysine (Provitro, 0413). For stimulation experiments, astrocytes were activated for 4 hours by stimulation with 20 ng/mL TNF-α (R&D, 210-TA-005) and 20 ng/mL IL-1β (R&D, 201-LB-005), 20 ng/mL IFN-γ (R&D, 485-MI-100/CF), 20 ng/mL IL-6 (R&D, 406-ML-005/CF), or 20 ng/mL GM-CSF (R&D, 415-ML-005/CF). After 4 hours, modulators were administered in addition to fresh TNF-α (10 ng/mL) and IL-1β (10 ng/mL), 20 ng/mL IFN-γ (R&D, 485-MI-100/CF), 20 ng/mL IL-6 (R&D, 406-ML-005/CF), or 20 ng/mL GM-CSF (R&D, 415-ML-005/CF) at the following concentrations: afatinib (Tocris, 6812), 10 μM; pemigatinib (Selleck Chemicals, S0088), 10 μM; and UNC2025 (Selleck Chemicals, S9662), 10 μM.

Linnerbauer M., Lößlein L., Vandrey O., Tsaktanis T., Beer A., Naumann U.J., Panier F., Beyer T., Nirschl L., Kuramatsu J.B., Winkler J., Quintana F.J, & Rothhammer V. (2022). Intranasal delivery of a small-molecule ErbB inhibitor promotes recovery from acute and late-stage CNS inflammation. JCI Insight, 7(7), e154824.

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Generating PH_AKT-TagRFP-T Plasmids

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To generate PH_AKT–TagRFP-T plasmids, DNA sequences for wildtype and R25C mutant of PH_AKT from PH-Akt-GFP plasmid (#51465, Addgene) and PH-Akt(R25C)-GFP plasmid (#51466, Addgene) were amplified by PCR and cloned into pmTagRFP-T-N1 (50 (link)) using XhoI and BamHI sites. PH_AKT–stagRFP was generated by site-directed mutagenesis of TagRFP-T (D159V) in the PH_AKT–TagRFP-T plasmid. Exo70(K632A, K635A)-CRY2-mCherry, CIB1-CAAX, and TfRc-pH were previously described (16 (link)). All cloning was done using standard molecular biology techniques. Es2 was purchased from Sigma or Cayman Chemical and stored as a 200-mM stock solution in DMSO at −30°C. Wortmannin, gefitinib, and afatinib were purchased from Tocris Bioscience and stored as 10 mM, 100 mM, and 20 mM stock solutions, respectively, in DMSO at −30°C.

An S.J., Anneken A., Xi Z., Choi C., Schlessinger J, & Toomre D. (2022). Regulation of EGF-stimulated activation of the PI-3K/AKT pathway by exocyst-mediated exocytosis. Proceedings of the National Academy of Sciences of the United States of America, 119(48), e2208947119.

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Cell Line Characterization and MUC1 Quantification

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Detailed description can be found in Supplemental Materials and Methods for: cell line cultures conditions; antibodies and gRNA sequences used; MS instrument settings; in vivo PET imaging Cell lines, SILAC labeling, and reagents Human breast cancer cell lines SKBR3, BT474, BT549, and MDA-MB-231; human non-small cell lung cancer cell lines H292 and HCC827; and HEK-293T were obtained from American Type Culture Collection (ATCC). SUM149 cells were a gift from Prof. W.T.A. van der Graaf (Medical Oncology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands). Stable isotope labeling of cell lines (SILAC) was done using RPMI or DMEM-high glucose media with normal Arg and Lys (light) or Arg 10 and Lys 8 (heavy) (Silantes). Human MUC1 shedding was measured using a standardized MUC1 human ELISA kit (EHMUC1; Thermo Scientific), mouse MUC1 was measured using a MUC1 mouse ELISA kit (E-EL-M2604; Elabscience). MUC1 has a reported serum half-life of ~7 days 47 . The following inhibitors were used: EGFR inhibitors erlotinib (LC Laboratories; Axon Medchem), gefitinib (Axon Medchem), lapatinib (LC Laboratories), afatinib (Tocris); JAK2 inhibitor BMS-911543 (Selleckchem); PI3K inhibitor BEZ235, AKT inhibitor MK2206, mTORC1/2 inhibitor everolimus, ERK inhibitor FR18024, MEK1 inhibitor AZD6244 (Axon Medchem).

de Boer H.R., Pool M., Joosten E., Everts M., Samplonius D.F., Helfrich W., Groen H.J.M., van Cooten S., Fusetti F., Fehrmann R.S.N., de Vries E.G.E, & van Vugt M.A.T.M. (2019). Quantitative proteomics analysis identifies MUC1 as an effect sensor of EGFR inhibition. Oncogene, 38(9).

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Plasmid Generation for Optogenetic Tools

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To generate PHAKT-TagRFP-T plasmids, DNA sequences for wildtype and R25C mutant of Exo70(K632A, K635A)-CRY2-mCherry, CIB1-CAAX and TfRc-pH were previously described (An et al., 2021) . All cloning was done using standard molecular biology techniques. Endosidin2 (Es2) was purchased from Sigma or Cayman Chemical and stored as a 200 mM stock solution in DMSO at -30 ºC. Wortmannin, gefitinib and afatinib were purchased from Tocris Bioscience and stored as 10 mM, 100 mM and 20 mM stock solutions, respectively, in DMSO at -30 ºC.

An S.J., Anneken A., Xi Z., Choi C.G., Schlessinger J., & Toomre D. (2022). Regulation of EGF-stimulated activation of the PI-3K/AKT pathway by exocyst-mediated exocytosis.

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