Human ABCG2, containing an amino (N)-terminal Flag tag, was expressed in HEK293-EBNA (Thermo Fisher Scientific) cells and purified as described previously [13 (link), 14 (link)].
Hek293 ebna
Manufactured by Thermo Fisher Scientific
Sourced in United States
HEK293-EBNA is a cell line derived from human embryonic kidney cells that stably express the Epstein-Barr virus nuclear antigen (EBNA). This cell line is commonly used for the production of recombinant proteins and viral vectors in research and development applications.
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Lab products found in correlation
Anti flag m2 affinity agarose gel, by Merck Group (3 mentions)
Superdex 200 increase 10 300 column, by GE Healthcare (2 mentions)
Cholesteryl hemisuccinate chs, by Anatrace (2 mentions)
Flag peptide, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Dnase 1, by Roche (2 mentions)
A673 cells, by American Type Culture Collection (2 mentions)
Superdex 200 10 300 column, by GE Healthcare (1 mentions)
Mkn45, by Thermo Fisher Scientific (1 mentions)
Mkn 45, by Leibniz Institute DSMZ (1 mentions)
Mkn74, by Thermo Fisher Scientific (1 mentions)
Lipofectamine ltx, by Thermo Fisher Scientific (1 mentions)
Fcs gold, by GE Healthcare (1 mentions)
Huvecs, by Thermo Fisher Scientific (1 mentions)
Arpe 19 cell, by American Type Culture Collection (1 mentions)
Opti mem 1, by Thermo Fisher Scientific (1 mentions)
Mcf 7, by American Type Culture Collection (1 mentions)
Sk n mc, by American Type Culture Collection (1 mentions)
11 protocols using hek293 ebna
1
Expression and Purification of Flag-tagged ABCG2
2
Purification of Flag-tagged Human ABCG2 from HEK293 Cells
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Human wild type ABCG2 (Uniprot: Q9UNQ0) or ABCG2 R184A containing an N-terminal Flag-tag was expressed in HEK293-EBNA (Thermo Fisher Scientific) cells by transient transfection19 (link). Cells were incubated at 37°C for 48–60 h before harvesting. Harvested Cell were lysed using a Dounce homogenizer and solubilized with 1% DDM, 0.1% CHS (cholesteryl hemisuccinate) (w/v) (Anatrace), 40 mM HEPES buffer pH 7.5, 150 mM NaCl, 10% (v/v) glycerol, 1 mM PMSF (phenylmethylsulfonyl fluoride), 2 μg ml−1 DNaseI (Roche), and protease inhibitor cocktail (Sigma). Lysed cells were centrifuged at 100,000 g and the supernatant was incubated with anti-Flag M2 affinity agarose gel (Sigma). ABCG2 was eluted with Flag peptide (Sigma) and applied to a Superdex 200 increase 10/300 column (GE Healthcare) in 40 mM HEPES, pH 7.5, 150 mM NaCl, 0.026% DDM and 0.0026% CHS (w/v). Peak fractions were collected for further use.
3
Expression and Purification of Flag-tagged ABCG2
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Human ABCG2, containing an amino (N)-terminal Flag tag, was expressed in HEK293-EBNA (Thermo Fisher Scientific) cells and purified as described previously [13 (link), 14 (link)].
4
ABCG2 Transporter Purification Protocol
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Full-length FLAG-ABCG2 from Homo sapiens was expressed in HEK293-EBNA (Thermo Fisher Scientific) cells by transient transfection (13 (link)). Cells were cultured at 37 °C under 8% CO2 and harvested 48 to 60 h after transfection. The collected cells were resuspended in buffer A (40 mM HEPES, pH 7.5, and 150 mM NaCl) with 10% glycerol, 1 mM PMSF (phenylmethylsulfonyl fluoride), 2 μg/mL DNaseI (Roche), and protease inhibitor cocktail (Sigma) and lysed using a Dounce homogenizer. Cell lysates were further solubilized with 1% DDM and 0.1% (w/v) CHS (cholesteryl hemisuccinate, Anatrace). After solubilization, cell lysates were spun at 100,000 g, and the supernatant was incubated with anti-Flag M2 affinity agarose gel (Sigma). Proteins were eluted by flag peptide and concentrated for gel-filtration purification with the Superdex 200 Increase 10/300 column (GE Healthcare) in buffer A with 0.026% DDM and 0.0026% CHS (w/v). Fresh peaks were collected for further experiments.
5
ABCG2 Protein Expression and Purification
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Expression and purification of human ABCG2. Human ABCG2, containing an amino (N)-terminal Flag tag, was expressed in HEK293-EBNA (Thermo Fisher Scientific) cells [52] and purified as described previously [29] . After transfection the cells were solubilized with 1% DDM (n-dodecyl-β-d-maltopyranoside) + 0.1% CHS (cholesteryl hemisuccinate) (w/v) (Anatrace), ultracentrifuged at 100,000g and the supernatant was then incubated with anti-Flag M2 affinity agarose gel (Sigma). ABCG2 was eluted with Flag peptide (Sigma), loaded into a Superdex 200 10/300 column (GE Healthcare) pre-equilibrated with 40 mM HEPES pH 7.5, 150 mM NaCl, 0.026% DDM + 0.0026% CHS (w/v), and the peak fractions collected.
6
Stable Troy Expression in Gastric Cell Lines
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The human gastric cell lines MKN45 (German Collection of Microorganisms and Cell Cultures, DSMZ, Braunschweig, Germany) and MKN74 (Japanese Health Science Research Resource Bank, Osaka, Japan) were cultured in RPMI supplemented with 20% or 10% fetal calf serum (FCS GOLD; PAA Laboratories, Pasching, Austria). HEK293ebna (Invitrogen, Carlsbad, USA) were maintained in Dulbecco's modified Eagles Medium (DMEM, 10% FCS). For generation of stable Troy expressing cell lines, MKN45 and MKN74 were transfected using Lipofectamine LTX (Invitrogen) and cultured under selection pressure with 650 μg/ml (MKN45) or 450 μg/ ml (MKN74) G418 (PAA) as described previously [33 ]. Overexpression was verified by qRT-PCR, immuncytochemistry and Western blotting (Supplementary Figure 1 ). For MKN45 Wnt signaling was induced at day 0 and 2 by incubating cell cultures with the recombinant proteins Wnt3a (BioCat, Heidelberg, Germany), RSPO1 (LifeTechnologies) or RSPO2 (antibodies-online) and analyzed at day 4. For demethylation studies, subconfluent cell cultures were daily treated with 1 μM 5-Aza-2’-Deoxycytidine (5azaC; BioCat) and expression profile was analyzed after 72 h.
7
HEK293, HUVEC, and ARPE-19 Cell Cultivation
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Cultivation of HEK293-EBNA (Invitrogen, Waltham, MA, USA), HUVECs (Life Technologies, Carlsbad, CA, USA) and ARPE-19 cells (American Type Culture Collection, Manassas, VA, USA) was performed as described in Ref. [8 (link)] and includes descriptions of media and media supplements.
8
Cell Line Culture Protocols
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The human embryonic kidney epithelial cell line HEK293 expressing the Epstein-Barr nuclear antigen 1, HEK293-EBNA (HEK293 hereon) was obtained from Invitrogen (Carlsbad, CA) and cultured in DMEM supplemented with 10% fetal bovine serum (FBS). The human M17 neuroblastoma cells (kind gift from Dr. Sic-Lung Chan, US Food and Drug Administration, Silver Spring, MD) were cultured in OPTI-MEM I (Invitrogen) supplemented with 10% FBS. The source and culturing conditions for the human breast cancer cell line MDA-MB-231 and JIMT-1 were described previously [1 (link), 41 (link)]. The human breast cancer cell lines SK-BR-3(SKBR3), BT-474, and MCF-7 were obtained from the American Type Culture Collection (Manassas, VA), and cultured in the same conditions as those for MDA-MB-231 and JIMT-1.
9
Cellular Assays for Oncogene Screening
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HEK-293EBNA (Invitrogen) and A673 cells (ATCC) were grown, retroviruses produced and used for infection, and soft agar assays were performed as described [24 (link), 28 (link), 37 (link)]. STR profiling and mycoplasma testing are performed annually on all cell lines. Dual luciferase reporter assays were performed in HEK-293EBNA cells as previously described [24 (link)]. 3.75–5.0 microgram of cDNA constructs were transfected into HEK-293EBNA cells and collected 48 h later for RNA-sequencing analysis.
10
Cell Line Characterization Protocol
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HEK-293 EBNA (human female; Invitrogen), A673 (human, female; ATCC, CRL-1598), SK-N-MC (human, female; ATCC, HTB-10), TC71 (human, male) and TTC-466 (human, female; T. Triche Laboratory, Keck School of Medicine of USC, Los Angeles, CA, USA), EWS-502 (human, sex unspecified; J.A. Fletcher Laboratory, Brigham & Women's Hospital, Boston, MA, USA).
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