Muc1 test was purchased from Thermo Scientific (EHMUC1). A total of 1 × 107 cells of each type were harvested and washed 3x with PBS. Next, the cells were suspended in 400 μL of a lysis buffer made of 50 mM Tris buffer, 100 mM NaCl, and x1 of protease inhibitor cocktail (Thermo-Scientific cat.#7448). Protein extraction was performed by sonication using the QSONICA Q500 sonicator. Six five-second sonications were followed by a 30 s cooldown time. Sonication was performed on ice. Protein extract was centrifuged at 15,000 rpm (21,130× g) for 30 min at 4 °C. The supernatant was collected and stored on ice. Protein content was tested using the Bradford protocol. ELISA test was performed according to the manufacturer’s guidelines.
Ehmuc1
Manufactured by Thermo Fisher Scientific
The EHMUC1 is a laboratory equipment designed for measuring various parameters. It provides accurate and reliable data for research and analysis purposes. The core function of the EHMUC1 is to perform measurements and data collection, enabling users to obtain essential information for their studies or experiments.
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Lapatinib, by LC Laboratories (1 mentions)
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Mk2206, by Axon Medchem (1 mentions)
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2 protocols using ehmuc1
1
Quantification of Muc1 Protein Expression
2
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).
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