Murine immortalized NMuMG cells (ATCC, no. CRL-1636) were cultured in DMEM plus 10% FBS and 10 μg/ml bovine insulin (Sigma-Aldrich). NMuMG cells stably over-expressing EPR (NMuMG-EPR) as well as cells stably transfected with the empty vector (mock) have been previously described (13 (link)) and were maintained in selective medium containing 800 μg/ml G418 (Sigma-Aldrich). Both wild-type NMuMG and NMuMG-EPR cells were transiently transfected with either control siRNAs or siRNA designed to knockdown murine EPR (5′-GAGCAAAAGAGAAUGCUUA-3′) (Thermo Fisher) using the Lipofectamine 2000 (Thermo-Fisher). NMuMG-EPR were transiently transfected with either control siRNA or with endoribonuclease-prepared esiRNA designed to knockdown murine Arrdc3 (EMU189761 from Sigma-Aldrich) using the Lipofectamine 2000 (Thermo-Fisher). For some experiments NMuMG and NMuMG-EPR cells were maintained in DMEM supplemented with 2% for 16 h prior to the addition of 5 ng/ml human recombinant TGF-β1 purchased from R&D Systems. SB431542 compound was purchased from Sigma-Aldrich, dissolved in Dimethyl sulfoxide (DMSO), and used at a 1 μM concentration.
Sb431542 compound
Manufactured by Merck Group
SB431542 is a small molecule compound that acts as a selective and potent inhibitor of the transforming growth factor-beta (TGF-β) type I receptor kinases ALK4, ALK5, and ALK7. It is commonly used as a research tool in cell and molecular biology studies.
Automatically generated - may contain errors
Lab products found in correlation
Bovine insulin, by Merck Group (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Tgf β1, by R&D Systems (1 mentions)
Recombinant human tgf β1, by R&D Systems (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Cycloheximide (chx), by Merck Group (1 mentions)
Mda mb 231, by Leibniz Institute DSMZ (1 mentions)
2 protocols using sb431542 compound
1
Murine NMuMG Cell Culture and Transfection
2
Culturing Murine and Human Mammary Cell Lines
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Murine immortalized NMuMG cells (ATCC, no. CRL-1636) were cultured in Dulbecco's Modified Eagle Medium (DMEM) plus 10% Fetal Bovine Serum (FBS) and 10 μg ml−1 bovine insulin (Sigma-Aldrich), 4T1 mouse mammary gland cancer cells (obtained from ATCC, no. CRL-2539) were cultured in DMEM/F12 plus 10% FBS, human mammary gland adenocarcinoma cells MDA-MB-231 (obtained from DSMZ, Germany, through Dr. G. Fronza, authenticated by STR DNA profiling) were cultured in DMEM plus 5% FBS, and human HEK-293 cells (ATCC, no. CRL-1573) were cultured in DMEM plus 10% FBS. HEK-293 cells were used to verify transient expression of FLAG-tagged EPRp based on their highly efficient transfectability. NMuMG cells were maintained in DMEM supplemented with 2% for 16 h prior to the addition of 1–10 ng ml−1 human recombinant TGF-β1 purchased from R&D Systems. All cell lines were tested for mycoplasma contamination and resulted negative. SB431542 compound was purchased from Sigma-Aldrich, dissolved in Dimethyl sulfoxide (DMSO), and used at a 1 μM concentration. Cycloheximide, dissolved in DMSO, was purchased from Sigma-Aldrich and used at a 5 μM concentration.
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