As described (9 (link), 10 (link), 16 (link)), breast cancer cell lines were grown to log-phase, washed twice with ice-cold PBS, and lysed in SDS lysis buffer according to the manufacturer's protocol (Cell Signaling Technology, Danvers, MA). Protein concentration was quantified using the bicinchoninic acid reagent (Sigma). Cell lysates containing 25 μg of protein were separated by SDS-polyacrylamide gel electrophoresis (PAGE), and transferred to PVDF membranes (Millipore). Membranes were immunoblotted with antibodies against phospho-AKT (S473), total AKT, phospho-p70S6K (T389), total p70S6K, phospho-rpS6 (S235/ 236), total rpS6, phospho-ERK (T202/Y204), total ERK (all from Cell Signaling Technology), tubulin (Sigma), and actin (Millipore). Bound antibody was visualized using SuperSignal West Pico (Thermo Scientific, Waltham, MA) or ECL plus (GE Healthcare, Auckland, NZ) and the chemiluminescence detection system by Fujifilm Las-3000.
Bicinchoninic acid reagent
Manufactured by Merck Group
Bicinchoninic acid reagent is a colorimetric assay used for the quantitative determination of total protein concentration in a sample. It works by the reduction of copper ions (Cu2+ to Cu1+) by proteins in an alkaline medium, and the subsequent chelation of the cuprous cation (Cu1+) by bicinchoninic acid to form a purple-colored complex that absorbs light at 562 nm.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (6 mentions)
Sds lysis buffer, by Cell Signaling Technology (5 mentions)
Tubulin, by Merck Group (4 mentions)
Supersignal west pico, by Thermo Fisher Scientific (4 mentions)
Ecl plus, by GE Healthcare (4 mentions)
Las 3000, by Fujifilm (4 mentions)
Actin, by Merck Group (3 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (2 mentions)
Chemidoc imaging system, by Bio-Rad (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Phospho rps6 s235 236, by Cell Signaling Technology (1 mentions)
Ab171380, by Abcam (1 mentions)
Ripa reagent, by Cell Signaling Technology (1 mentions)
Ecl detection kit, by Thermo Fisher Scientific (1 mentions)
Phospho p70s6k, by Cell Signaling Technology (1 mentions)
Hrp conjugated goat anti rabbit igg secondary antibody, by Thermo Fisher Scientific (1 mentions)
Protease phosphatase inhibitors cocktail, by Cell Signaling Technology (1 mentions)
Tetramethylethylenediamine, by Merck Group (1 mentions)
Polyvinylidene difluoride membrane, by Thermo Fisher Scientific (1 mentions)
Crystal violet, by Merck Group (1 mentions)
10 protocols using bicinchoninic acid reagent
1
Western Blot Analysis of Breast Cancer Cell Signaling
2
Western Blot Analysis of SOX2 in Breast Cancer Cells
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As described in detail previously (10 (link)), breast cancer cell lines were grown to log-phase, washed twice with ice-cold PBS, and lysed in sodium dodecyl sulfate (SDS) lysis buffer according to the manufacturer’s protocol (Cell Signaling Technology, Danvers, MA, USA). Protein concentration was quantified using the bicinchoninic acid reagent (Sigma). Cell lysates containing 25 µg of protein were separated by SDS-polyacrylamide gel electrophoresis, and transferred to PVDF membranes (Millipore). Membranes were immunoblotted with antibodies against SOX2 (Cell Signaling Technology) and tubulin (Sigma). Bound antibody was visualized using SuperSignal West Pico (Thermo Scientific, Waltham, MA) or ECL plus (GE Healthcare, Auckland, New Zealand) and the chemiluminescence detection system by Fujifilm Las-3000.
3
Western Blot Analysis of SOX2 in Melanoma
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Cell lysates of melanoma cells (M14 and A375) were prepared using the RIPA Reagent (Cell Signaling Technology, Inc.). The protein concentration was quantified with bicinchoninic acid reagent (Sigma-Aldrich; Merck KGaA). A total of 30 µg protein samples were fractionated on 15% SDS-polyacrylamide gels and then transferred onto PVDF membranes. Thereafter, the membranes were blocked with 5% skimmed milk at room temperature for 2 h and probed with primary antibodies targeting SOX2 (1:1,000, cat. no. cat. no. ab171380; Abcam) or β-actin (1:1,000; cat. no. ab8227; Abcam) at 4˚C overnight. Subsequently, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies (1:1,000; cat. no. ab205718, Abcam) at room temperature for 2 h. The bands were visualized using an ECL detection kit (Thermo Fisher Scientific, Inc.) and quantified using the ImageJ 1.8.0 software (National Institute of Health). The aforementioned antibodies used for this investigation were purchased from Abcam (Cambridge, MA, USA).
4
Western Blot Analysis of Breast Cancer Cells
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Breast cancer cell lines were grown to log-phase, washed twice with ice-cold PBS, and lysed in SDS lysis buffer according to the manufacturer’s protocol (Cell Signaling Technology, Danvers, MA). Protein concentration was quantified using the bicinchoninic acid reagent (Sigma). Cell lysates containing 25 µg of protein were separated by SDS-PAGE gel electrophoresis, and transferred to PVDF membranes (Millipore). Membranes were immunoblotted with antibodies against phospho-Akt (S473), total Akt, phospho-p70S6K (T389), total p70S6K, phospho-rpS6 (S235/236), total rpS6, phospho-ERK (T202/Y204), total ERK, (all from Cell Signaling Technology), tubulin (Sigma), and actin (Millipore). Bound antibody was visualized using SuperSignal West Pico (Thermo Scientific, Waltham, MA) or ECL plus (GE Healthcare, Auckland, NZ) and the chemiluminescence detection system by Fujifilm Las-3000. Densitometry was performed using ImageJ. The relative intensity of phosphorylated proteins was normalized using either tubulin or actin as the standard. The fold change was then calculated between MCF7 and BT20 as standard reference.
5
Western Blot Analysis of TfR1 Protein
6
Quantification of IDO1 Expression in Breast Cancer Cells
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This procedure was carried out as described previously [69 (link),71 (link),72 (link)]. Breast cancer cell lines were grown to log-phase, washed twice with ice-cold PBS, and lysed in a SDS lysis buffer (60 mM Tris-HCl (pH 6.8 at 25 °C), 2% (w/v) SDS, 10% glycerol). Protein concentration was quantified using the bicinchoninic acid reagent (Sigma). Proteins (25 μg) were separated by SDS-polyacrylamide gel electrophoresis (PAGE), and transferred to PVDF membranes (Millipore Merck, Auckland, New Zealand). Membranes were immunoblotted with antibodies against human IDO1 and tubulin (Sigma). Bound antibody was visualized using Pierce™ ECL Western Blotting Substrate (ThermoFisher Scientific) and the chemiluminescence measured using image analyser LAS-3000 (Fujifilm, Tokyo, Japan).
7
Western Blot Analysis of Breast Cancer Cells
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As described in detail previously [44 (link)], breast cancer cell lines were grown to log-phase, washed twice with ice-cold PBS, and lysed in SDS lysis buffer according to the manufacturer’s protocol (Cell Signaling Technology, Danvers, MA, USA). Protein concentration was quantified using the bicinchoninic acid reagent (Sigma). Cell lysates containing 25 µg of protein were separated by SDS–polyacrylamide gel electrophoresis (PAGE) and transferred to PVDF membranes (Millipore). Membranes were subjected to immunoblotting with antibodies against TDP1 (Abcam AB4166), PARP1 (Cell Signaling Technology #9542), and tubulin (Sigma). Bound antibody was visualized using the Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific) and chemiluminescence detection by the ChemiDoc imaging system (Bio-Rad).
8
Western Blot Analysis of Breast Cancer Cell Lines
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As described in detail previously [20 (link)], breast cancer cell lines were grown to log-phase, washed twice with ice-cold PBS, and lysed in SDS lysis buffer according to the manufacturer’s protocol (Cell Signaling Technology, Danvers, MA). Protein concentration was quantified using the bicinchoninic acid reagent (Sigma). Cell lysates containing 25 μg of protein were separated by SDS-polyacrylamide gel electrophoresis (PAGE), and transferred to PVDF membranes (Millipore). Membranes were immunoblotted with antibodies against phospho-AKT (S473), total AKT, phospho-p70S6K (T389), total p70S6K, phospho-rpS6 (S235/236), total rpS6, phospho-4E-BP1 (T70), total 4E-BP1, phospho-ERK (T202/Y204), total ERK (all from Cell Signaling Technology), tubulin (Sigma), and actin (Millipore). Bound antibody was visualized using SuperSignal West Pico (Thermo Scientific, Waltham, MA) or ECL plus (GE Healthcare, Auckland, NZ) and the chemiluminescence detection system by Fujifilm Las-3000. Densitometry was performed using ImageJ. The relative intensities of phosphorylated proteins were normalized using either tubulin or actin as the standard. The fold change was then calculated between MCF7 and BT20 as standard reference.
9
Evaluation of Coronarin-D Bioactivity
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Acetonitrile, ethanol, water, paraformaldehyde, and DMSO were obtained from Merck (Darmstadt, Germany). Roswell Park Memorial Institute-1640, BSA, trypsin, streptomycin, DMEM, and penicillin were purchased from Himedia Chemicals Ltd (Mumbai, India). MTT reagent, 2′,7′-dichlo-rodihydrofluorescein diacetate (DCFH-DA) reagent, JC-1 dye, Hoechst 33242, propidium iodide (PI), RNase A, crystal violet, methylene blue, Coomassie Brilliant Blue R-250, N-acetyl-l -cysteine (NAC), SDS, Triton-X 100, Tris-base, glycine, acrylamide, bisacrylamide, ammonium persulfate, tetramethylethylenediamine, Tween-20, bicinchoninic acid reagent, and 2-mercaptoethanol were purchased from Sigma-Aldrich. All antibodies were procured from Abcam (Cam-bridge, MA, USA). Annexin V-fluorescein isothiocyanate (FITC) apoptosis kit and APO Direct kit were obtained from BD Biosciences (San Jose, CA, USA). Caspase-3 substrate (Ac-DEVD-AMC), caspase-8 substrate (Ac-IETD-AMC), and caspase-9 substrate (Ac-LEHD-AMC) were obtained from Cayman Chemicals. Polyvinylidene difluoride membrane was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Matrigel invasion chambers with 8 µm pore size inserts were obtained from BD Biosciences. The reference standards used, that is, coronarin-D (purity >99%) and coronarin-D methyl ether (purity >99%), were purchased from Wuhan ChemFaces Biochemical Co., Ltd (Wuhan, China).
10
Cell Lysis and Protein Quantification
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Cell extracts Cells were lysed with the cell lysis buffer from Cell Signaling Technology in the presence of the cOmplete™ ULTRA, EDTA-free Protease Inhibitor Cocktail and the PhosSTOP™ phosphatase inhibitor from Roche. After a brief sonication on ice and centrifugation at 4°C, the protein concentration in the supernatant was determined with the Bicinchoninic Acid reagent from Sigma.
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