Extracellular matrix proteins deposited by human NP cells were obtained following published procedures (Pei et al. 2011 (link)) with slight modification. In brief, human NP cells were cultured in complete growth medium until 80% confluence. L-ascorbic acid phosphate (50 μg/mL) was supplemented in media with additional culture for 9 days. Cell-free extracellular matrix was then obtained by incubating the cells with 0.5% Triton X-100 (3 mL) containing 20 mM ammonium hydroxide in DPBS for 30 min at 37°C. Supernatant was collected, centrifuged at 2000 rpm for 8 min, and stored at −70°C until use. Total protein concentration (0.31 mg/mL) of harvested NP deposited ECM mixture (referred to as “matrix” in the following text) was determined with Detergent-compatible (Dc) protein assay kit (Bio-Rad, Hercules, CA) according to the manufacturer's instruction.
Detergent compatible protein assay kit
Manufactured by Bio-Rad
Sourced in United States
The Detergent-compatible protein assay kit is a laboratory product designed to quantify the total protein content in a sample. It is compatible with the presence of detergents, allowing for accurate protein measurement in samples containing detergents. The kit provides a reliable and reproducible method for protein determination.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (6 mentions)
Miscript reverse transcription kit, by Qiagen (4 mentions)
Miscript sybr green pcr kit, by Qiagen (4 mentions)
Mirneasy mini kit, by Qiagen (4 mentions)
Rpmi 1640, by Thermo Fisher Scientific (4 mentions)
Complete protease inhibitor cocktail, by Roche (4 mentions)
Ros assay kit, by Nanjing Jiancheng (4 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
Protease inhibitor cocktail, by Merck Group (3 mentions)
Sodium molybdate and protease inhibitors, by Cell Signaling Technology (3 mentions)
Ethylene glycol tetraacetic acid (egta), by Research Products International (3 mentions)
Oasis hlb cartridge, by Waters Corporation (2 mentions)
Phosstop phosphatase inhibitor cocktail, by Roche (2 mentions)
Trypsin, by Roche (2 mentions)
Anti mouse cd4 fluorescein isothiocyanate fitc, by Thermo Fisher Scientific (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (2 mentions)
Las 4000 luminescent image analyzer, by GE Healthcare (2 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (2 mentions)
76 protocols using detergent compatible protein assay kit
1
Extraction of NP-Derived Extracellular Matrix
2
Purification and Detection of Epitope-Tagged Proteins
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Tween 20, Triton X-100 (TX-100), and Triton X-114 (TX-114) were obtained from Sigma-Aldrich (St. Louis, MO). Strep-tag and Strep-Tactin XT were purchased from IBA Lifesciences (Gottingen, Germany). A detergent-compatible (DC) protein assay kit was bought from Bio-Rad Laboratories (Hercules, CA). Anti-FLAG epitope (DYKDDDDK ; catalog no. 637301) and horseradish peroxidase (HRP)-conjugated donkey anti-human IgG antibody (catalog no. 410902) were purchased from Biolegend (San Diego, CA). The secondary antibody anti-rat IgG-HRP (catalog no. 7077) was bought from Cell Signaling Technology (CST [Danvers, MA]). All other reagents and chemicals, including nickel-nitrilotriacetic acid (Ni-NTA) agarose and the Pierce Rapid Gold BCA (bicinchoninic acid) protein assay kit, were purchased from Fisher Scientific International, Inc., (Hampton, NH) and were of the highest purity or analytical grade commercially available.
3
Establishing Cell Culture and Cytokine Assays
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Ammonium sulfate [(NH4)2SO4] was purchased from Merck, Darmstadt,
Germany. Bovine serum albumin, lipopolysaccharide (LPS), phosphate
buffer saline (PBS), and streptozotocin (STZ) were purchased from
Sigma-Aldrich Co., St. Louis, Missouri, USA. Dulbecco’s modified
Eagle’s medium (DMEM), fetal bovine serum (FBS) 0.25% trypsin/0.01
M ethylenediaminetetraacetic acid, and penicillin/streptomycin solution
were purchased from Gibco, Invitrogen, USA. The 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) assay kit was purchased from
Boehringer Mannheim, Mannheim, Germany. A tumor necrosis factor-α
(TNF-α) enzyme-linked immunosorbent assay (ELISA) kit was purchased
from eBioscience, Inc., California, USA. A detergent compatible (DC)
protein assay kit was obtained from Bio-Rad Laboratories, Philadelphia,
USA.d -Glucose, di-sodium hydrogen phosphate (Na2HPO4), citric acid, and sodium hydroxide (NaOH) were purchased
from Ajax Finechem, New South Wales, Australia. Peptone and yeast
extract were obtained from HiMedia Laboratories, Maharashtra, India.
Hematoxylin and eosin staining dyes were obtained from C.V. Laboratories
Co., Ltd., Bangkok, Thailand. Aerrene isoflurane, USP, was purchased
from Baxter, USA.
Germany. Bovine serum albumin, lipopolysaccharide (LPS), phosphate
buffer saline (PBS), and streptozotocin (STZ) were purchased from
Sigma-Aldrich Co., St. Louis, Missouri, USA. Dulbecco’s modified
Eagle’s medium (DMEM), fetal bovine serum (FBS) 0.25% trypsin/0.01
M ethylenediaminetetraacetic acid, and penicillin/streptomycin solution
were purchased from Gibco, Invitrogen, USA. The 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) assay kit was purchased from
Boehringer Mannheim, Mannheim, Germany. A tumor necrosis factor-α
(TNF-α) enzyme-linked immunosorbent assay (ELISA) kit was purchased
from eBioscience, Inc., California, USA. A detergent compatible (DC)
protein assay kit was obtained from Bio-Rad Laboratories, Philadelphia,
USA.
from Ajax Finechem, New South Wales, Australia. Peptone and yeast
extract were obtained from HiMedia Laboratories, Maharashtra, India.
Hematoxylin and eosin staining dyes were obtained from C.V. Laboratories
Co., Ltd., Bangkok, Thailand. Aerrene isoflurane, USP, was purchased
from Baxter, USA.
4
Uric Acid Treatment of Insulin-Producing Cells
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All chemical reagents were purchased from Sigma (St. Louis, MO, US) unless otherwise stated. Min6 cells were maintained in 25 mmol/L glucose Dulbecco's Modified Eagle's Medium (DMEM, Hyclone, Logan, UT, USA), and supplemented with 10% fetal bovine serum (FBS), 50 μmol/L β-mercaptoethanol, 100 U/mL penicillin and 0.1 mg/mL streptomycin in 5% CO2 at 37°C. For cell treatments, uric acid solution was prepared as previously described[12] (link). Antibodies to iNOS and MafA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The detergent compatible (DC) protein assay kit was purchased from Bio-Rad Laboratories (Hercules, CA, USA). Recombinant human uric acid was purchased from R&D System (Minneapolis, MN, USA).
5
Investigating Autophagy Modulation by H2S
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Ham’s F12 medium was obtained from Sigma-Aldrich (St. Louis, USA). Fetal bovine serum (FBS) was from Hyclone (Logan, UT, USA). Lipofectamine 2000 transfection reagent was obtained from Invitrogen Life Technologies (Grand Island, NY, USA). NaHS was purchased from Sigma Aldrich (St. Louis, MO). Antibodies against Beclin-1, LC3-I, LC3-II and β-Actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The Detergent Compatible (DC) Protein Assay kit was purchased from Bio-Rad Laboratories (Hercules, CA, USA).
6
PGE2 Signaling Pathway Analysis
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Dulbecco-modified Eagle medium (DMEM) and Lipofectamine 2000 transfection reagent were obtained from Invitrogen Life Technologies (Grand Island, NY, USA). Fetal bovine serum (FBS) was purchased from GIBCO (Burlington, ON, USA). ART was purchased from Bide Pharmaceutical Corporation (Guangzhou, China). PGE2 was purchased from Sigma Aldrich (St Louis, MO, USA). The human PGE2 ELISA kit was obtained from Uscn Life Science Inc (Wuhan, China). Antibodies against COX-2 and β-Actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The Detergent Compatible (DC) Protein Assay kit was purchased from Bio-Rad Laboratories (Hercules, CA, USA).
7
Celastrol Modulates mTOR Signaling
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Dulbecco’s modified Eagle’s medium (DMEM), sodium pyruvate were from Gibco-BRL (Rockville, MD, USA). Fetal bovine serum (FBS) was from PAA Laboratories (GmbH, Linz, Austria). Celastrol (purity > 98 %) was purchased from Alexis Biochemicals (San Diego, CA, USA). Leucine (purity 99 %) and dimethylthiazoly-2, 5-diphenyltetrazolium bromide (MTT) were obtained from Sigma Aldrich. Antibodies against mTOR, p-mTOR, p-S6, p-p70S6k, and p-BAD were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-Rictor, anti-p27 and anti-β-actin antibodies were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA, USA). K-LISA™ mTOR activity kit was obtained from Millipore (Boston, MA, USA). The Detergent Compatible (DC) Protein Assay kit was purchased from Bio-Rad Laboratories (Hercules, CA, USA). The miRNeasy Mini kit, the miScript Reverse Transcription kit and the miScript SYBR Green PCR kit were purchased from Qiagen (Hilden, Germany).
8
Isobaric Labeling and Phosphoproteomic Analysis
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A tandem mass tag (TMT) 10-plex isobaric label reagent set and Lithium dodecyl sulfate (LDS) sample buffer were purchased from Thermo Fisher Scientific (Waltham, MA). The PhosSTOP™ phosphatase inhibitor cocktail, cOmplete™ protease inhibitor cocktail, and Trypsin were obtained from Roche (Basel, Switzerland). Phosphate-buffered saline tablets were purchased from Takara (Shiga, Japan). A detergent-compatible (DC) protein assay kit was obtained from Bio-Rad (Hercules, CA). XV Pantera gradient gel (5 to 20%) was purchased from DRC (Tokyo, Japan). An ImmunoStar LD kit and Lysyl Endopeptidase (Lys-C) were obtained from Wako (Osaka, Japan). Oasis HLB cartridges were purchased from Waters (Milford, MA).
ATM (D2E2), pCHEK1 S345 (133D3), and pCHEK2 T68 (C13C1) antibodies were obtained from Cell Signaling Technology (Danvers, MA). CHEK1 (DCS-310) and CHEK2 (DCS-270) antibodies were obtained from MBL (Nagoya, Japan). pATM S1981 (10H11.E12) antibody was purchased from Merck (Burlington, MA). ATR (N-19) antibody was obtained from SantaCruz (Dallas, TX). α-Tubulin (DM1A) antibody was purchased from Thermo Fisher Scientific.
ATM (D2E2), pCHEK1 S345 (133D3), and pCHEK2 T68 (C13C1) antibodies were obtained from Cell Signaling Technology (Danvers, MA). CHEK1 (DCS-310) and CHEK2 (DCS-270) antibodies were obtained from MBL (Nagoya, Japan). pATM S1981 (10H11.E12) antibody was purchased from Merck (Burlington, MA). ATR (N-19) antibody was obtained from SantaCruz (Dallas, TX). α-Tubulin (DM1A) antibody was purchased from Thermo Fisher Scientific.
9
Cell Culture Reagents and Protocols
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Dispase, Cell Recovery Solution, Penicillin-streptomycin, and fetal bovine serum (FBS) were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Oasis HLB cartridges were purchased from Waters (Milford, MA, USA). A detergent-compatible (DC) protein assay kit was obtained from Bio-Rad (Hercules, CA, USA). Chemi-Lumi Super and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Nacalai Tesque (Kyoto, Japan). Phosphate-buffered saline (PBS) tablets were obtained from Takara (Shiga, Japan). cOmplete protease inhibitor cocktail, PhosSTOP phosphatase inhibitor cocktail, and trypsin were obtained from Roche (Basel, Switzerland).
10
Cytoprotective Effects of NaHS
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RPMI 1640 and DMEM were obtained from GIBCO (Invitrogen Company). Fetal bovine serum (FBS) was obtained from Hyclone (Logan, UT, USA). Lipofectamine 2000 transfection reagent was obtained from Invitrogen Life Technologies (Grand Island, NY, USA). NaHS was purchased from Sigma Aldrich (St. Louis, MO, USA). Antibodies against Nrf-2, GST, SOD, HO-1, and β-Actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The Detergent Compatible (DC) Protein Assay kit was purchased from Bio-Rad Laboratories (Hercules, CA, USA).
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