HNSCC cell lines SNU1041 and FaDu were obtained from Korea Cell Line Bank (Seoul, Korea) and maintained in Dulbecco's Modified Eagle's Medium (DMEM; Invitrogen, Carlsbad, CA, USA), 10% FBS (Invitrogen), and penicillin/streptomycin (Invitrogen). Primary sphere cells (K3, K4, and K5) were isolated from surgical specimens from HNSCC patients, and the CSC properties were validated using a number of functional assays testing the self-renewal capability, stem cell marker expression, chemoresistance, and in vivo tumorigenicity, as reported previously (Lim et al, 2011 (link)). Primary sphere cells expanded in serum-free DMEM Ham's F-12 (DMEM/F12) medium supplemented with human recombinant basic fibroblast growth factor (bFGF; 10 ng/ml; R&D Systems, Minneapolis, MN, USA), N2 supplement (GIBCO, Franklin Lakes, NJ, USA), and epidermal growth factor (EGF; 10 ng ml−1; R&D Systems). We purchased primary antibodies against proteins Oct4, Nanog, CD44, Snail, cyclin B1, vimentin, and ABCG2 from Santa Cruz Biotechnology (Santa Cruz, CA, USA), Ki-67 from Chemicon International Inc. (Temecula, CA, USA), SOX2 from Abcam (Cambridge, UK), E-cadherin from Cell Signalling Technology (Beverly, MA, USA), and secondary antibodies, anti-rabbit IgG and anti-mouse IgG, from Jackson ImmunoResearch Laboratories (West Grove, PA, USA).
Abcg2
Manufactured by Santa Cruz Biotechnology
Sourced in United States
ABCG2 is a membrane-bound transporter protein that is involved in the efflux of various substrates from cells. It plays a role in the regulation of cellular homeostasis and the protection of cells from toxic substances.
Automatically generated - may contain errors
Lab products found in correlation
E cadherin, by Cell Signaling Technology (2 mentions)
β actin, by Merck Group (2 mentions)
Nanog, by Cell Signaling Technology (2 mentions)
Quantity one software, by Bio-Rad (2 mentions)
Gapdh, by Merck Group (2 mentions)
P akt, by Cell Signaling Technology (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
N2 supplement, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Human recombinant basic fibroblast growth factor bfgf, by R&D Systems (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Epidermal growth factor (egf), by R&D Systems (1 mentions)
Vimentin, by Santa Cruz Biotechnology (1 mentions)
Nanog, by Santa Cruz Biotechnology (1 mentions)
Cyclin b1, by Santa Cruz Biotechnology (1 mentions)
Snu1041, by Korean Cell Line Bank (1 mentions)
Snail, by Santa Cruz Biotechnology (1 mentions)
β actin, by Abcam (1 mentions)
Abcb1, by Santa Cruz Biotechnology (1 mentions)
22 protocols using abcg2
1
Characterization of HNSCC Cancer Stem Cells
2
Western Blot Analysis of ABC Transporters
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Western blot analysis was performed as described (Izumchenko et al, 2009 (link)). Anti-Abcc10 antibody was used at a concentration of 1 : 500 (Hopper-Borge et al, 2011 (link)). ABCB1, ABCG2 (Santa Cruz Biotechnology, Dallas, TX, USA), β-actin (Abcam, Cambridge, MA, USA), HER2 and ERα (Cell Signalling Technology, Danvers, MA, USA) antibodies were used according to the manufacturer's instructions.
3
Western Blot Analysis of Stem Cells
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Samples were analyzed by western blotting as described previously.25 (link),55 (link) Cells were washed with ice-cold PBS, and total protein extract was prepared with RIPA buffer with 1× Halt protease inhibitor cocktail and Halt phosphatase inhibitor (Thermo Fisher Scientific) at a ratio of 5 × 106 cells/mL. 30 μg protein for iPSC-derived OCs or 15 μg for iPSC-LESCs was loaded onto 4%–15% Mini-PROTEAN TGX gels (Bio-Rad, CA, USA) and transferred to an Immun-Blot polyvinylidene fluoride (PVDF) membrane using a Trans-Blot SD semi-dry transfer cell (Bio-Rad). Membranes were blocked with 5% non-fat dry milk in PBS-Tween 20 (0.1%) for 2 h and incubated overnight at 4°C with the following primary antibodies diluted in blocking buffer: PAX6 (1:2,000, Covance), ABCG2 (1:1,000, Santa Cruz Biotechnology), and β-actin (1:5,000, Sigma-Aldrich). Incubation with horseradish peroxidase-conjugated secondary antibody (anti-mouse or -rabbit, 1:5,000, Applied Biosystems) was done for 2 h at room temperature. Membranes were incubated with Clarity Western ECL Substrate (Bio-Rad) and imaged using the ChemiDoc XRS Imaging System (Bio-Rad). Band intensities were quantified using the Fiji/ImageJ software (National Institutes of Health, MD, USA).
4
Molecular Mechanisms of 5-FU in Cancer
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The 5-FU (Sigma-Aldrich Inc., St. Louis, MO, USA) was diluted in dimethyl sulfoxide and stored at −20 °C. Primary antibodies against the following proteins were used: NRF2 (sc-365949; Santa Cruz Biotechnology Inc., CA, USA), HO-1 (sc-136960; Santa Cruz Biotechnology Inc., CA, USA), SOD2 (LF-PA0021; Ab Frontier, Seoul, Korea), NQO-1 (#3187; Cell Signaling Technology, MA, USA), P-P38 (#4511; Cell Signaling Technology, MA, USA), P38 (#8690; Cell Signaling Technology, MA, USA), E-cadherin (#3195; Cell Signaling Technology, MA, USA), N-cadherin (#13116; Cell Signaling Technology, MA, USA), ZEB-1 (#3396; Cell Signaling Technology, MA, USA), ABCG2 (sc-377176; Santa Cruz Biotechnology Inc., CA, USA), Nanog (#3580; Cell Signaling Technology, MA, USA), Oct4 (ab18976; Abcam, MA, USA), CD133 (MAB4310; Millipore Billerica, MA, USA), and glyceraldehyde 3-phosphate
Dehydrogenase (GAPDH) (sc-47724; Santa Cruz Biotechnology Inc., CA, USA). Horseradish peroxidases (HRP)-conjugated anti-rabbit and anti-mouse IgG antibodies were obtained from Millipore (AP132P, AP124P; Billerica, MA, USA).
Dehydrogenase (GAPDH) (sc-47724; Santa Cruz Biotechnology Inc., CA, USA). Horseradish peroxidases (HRP)-conjugated anti-rabbit and anti-mouse IgG antibodies were obtained from Millipore (AP132P, AP124P; Billerica, MA, USA).
5
Western Blot Analysis of Stem Cell Markers
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Western blots were performed as described previously 24 (link) using antibodies specific for Bmi1, SOD2, Slug (Cell Signaling Technology, Beverly, MA, USA), Nanog (Aviva Systems Biology, San Diego, CA, USA), C-myc, ABCG2 (ATP-binding cassette transporters) (Santa Cruz, Dallas, Texas, USA) and GAPDH (Sigma, San Louis, MO, USA). The results obtained from western blot were quantified by Quantity One software (Bio-Rad) and shown in Supplementary Material : Figure S2.
6
Western Blot Analysis of ABCG2, ERCC1, and Slug
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Western blots were performed as previously described [43 (link)] using antibodies specific for ABCG2 (Santa Cruz, CA, USA), ERCC1 (Atlas Antibodies, Stockholm, Sweden), and Slug (Cell Signaling Technology, Beverly, MA); GAPDH (Sigma-Aldrich, MO, USA) was included as a control. The results obtained from western blotting were quantified using Quantity One software (Bio-Rad, CA, USA); the results are shown in Electronic Supplementary Figure S1 .
7
Protein Expression Analysis of Stem Cells
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Spheres and parent cells were harvested and lysed with protein extraction buffer (0.5 M NaCl, 0.5 M Tris-HCl, 50 mM EDTA, 50 mM EGTA, 10% triton X-100, 1 mg sodium deoxycholate, 1 mM Na3VO4, 1mM phenylmethylsulfonyl fluoride, EDTA-free protease inhibitor, and distilled water). Proteins (20 μg) were separated on 6 - 10% SDS-PAGE, and transferred onto a nitrocellular membrane. After blocked with 5% skim milk solution in Tris-buffered saline with 0.1% Tween 20 (0.1% TBS-T), the membranes were incubated with primary antibodies specific for MDR1 (Cell Signaling Technology, Beverly, MA), ABCG2 (Santa Cruz Biotechnology, Santa Cruz, CA), total OXPHOS complexes (Abcam, Cambridge, MA), and PGC1α (Calbiochem, Darmstadt, Germany). Signals were visualized using a chemiluminescence detection kit (AbFrontier, Seoul, Korea).
8
Protein Expression Analysis by Western Blot
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Cells were lysed with RIPA buffer (Cell signaling technology, Danvers, MA), the proteins were fractionated by SDS-PAGE and western blotting was performed. The blots were incubated with diluted primary antibodies overnight at 4°C. Then, blots were exposed to the appropriate peroxidase-conjugated secondary antibodies (Cell Signaling Technology, MA) at 1:3000 for 2 h at room temperature. Finally blots were developed with Luminata Crescendo western HRP substrate (Millipore, MA). The following antibodies were used: EIF5A2 (1:1000) (Wolwo, Shanghai, China), Casepase-8 (1:1000), PARP (1:1000), CD44 (1:500), nanog (1:500) and p75NTR (1:500) (Cell Signaling Technology, MA), ABCG2 (1:500), CD24 (1:500), β-tubulin (1:1000) and actin (1:1000) (Santa Cruz Biotechnology, TX).
9
Immunofluorescence Staining of Nrf2 and ABCG2
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Cells were seeded in 12-well plates containing autoclaved glass coverslips and cultured in DMEM or RPMI-1640 medium containing 10% FBS. After thrice PBS washing, cells were covered with ice-cold 100% methanol for 10 min at −20 °C, and rinsed in PBS for 5 min. Then cells were blocked in Blocking Buffer (1 × PBS/5% normal serum/0.3% Triton™ X-100) for 60 min at room temperature, prior to incubating with primary antibody (Nrf2, 1:100; ABCG2, 1:50; Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4 °C. Finally, cells were rinsed thrice in PBS, and incubated in fluorochrome-conjugated secondary antibody diluted in Antibody Dilution Buffer (1 × PBS/1% BSA/0.3% Triton X-100) for 2 h at room temperature in dark. Nuclei were counterstained with 4,6- diamidino-2-phenylindole (DAPI). Confocal laserscanning microscopy was performed using a Leica TCS SP5 confocal microscope (Leica, Mannheim, Germany).
10
Modulating ABCG2 Sensitivity in Ovarian Cancer
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A2780/MLN-R human ovarian cancer cells were infected with lentiviral particles containing nontargeted (control) or targeted short hairpin RNA (shRNA) directed at ABCG2 (Santa Cruz Biotechnology) according to the manufacturer’s instructions. Positively infected cells were selected with puromycin treatment. Drug-induced cytotoxicity was quantified by MTT cell viability assay as described previously [38 (link)]. Knockdown efficiency was assessed by immunoblotting.
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