The antibodies against S100A8 and p62 were obtained from Santa Cruz Biotechnology (Sana Cruz, CA, USA). The antibodies to Actin, BECN1, PI3KC3, C-PARP, ULK1, Bcl-2 and P-ULK1 were from Cell Signaling Technology (Boston, MA, USA). The antibodies to LC3 and TLR-4 were purchased from Abcam (Cambridge, MA, USA). Anti-Atg7 antibody was from Novus (Denver-Littleton, CO, USA). Vincristine (VCR), adriamycin (ADM), rotenone (Rot), thenoyltrifluoroacetone (TTFA), antimycin A (AA), E64D, anti-RAGE antibody and pepstatin were from Sigma (Milpitas, CA, USA). Full-length human S100A8 cDNA (pLPCX-S100A8) was a gift from Dr. RW Stam (Erasmus Medical Center/Sophia Children's Hospital, Netherlands). FITC-Annexin V Apoptosis Detection kit and the Nuclear and Cytoplasmic Protein Extraction kit were purchased form Beyotime Institute of Biotechnology (Beijing, China). S100A8 protein was obtained from Novus Biologicals. Contaminating LPS was removed by Triton X-114 extraction. LPS content was always below 0.5 ng/mg protein, which did not cause an effect in our assays.
Thenoyltrifluoroacetone
Manufactured by Merck Group
Sourced in United States
Thenoyltrifluoroacetone is a chemical compound used in laboratory settings. It functions as a chelating agent, capable of forming stable complexes with various metal ions.
Automatically generated - may contain errors
Lab products found in correlation
Antimycin a, by Merck Group (3 mentions)
Oligomycin, by Merck Group (2 mentions)
Rotenone, by Merck Group (2 mentions)
Annexin 5 fitc apoptosis detection kit, by Beyotime (1 mentions)
Nuclear and cytoplasmic protein extraction kit, by Beyotime (1 mentions)
Pepstatin, by Merck Group (1 mentions)
C parp, by Cell Signaling Technology (1 mentions)
P ulk1, by Cell Signaling Technology (1 mentions)
Pi3kc3, by Cell Signaling Technology (1 mentions)
Bcl 2, by Cell Signaling Technology (1 mentions)
Becn1, by Cell Signaling Technology (1 mentions)
Adenosine triphosphate (atp), by Merck Group (1 mentions)
Digitonin, by Merck Group (1 mentions)
Itaconic acid, by Merck Group (1 mentions)
Phenazine methosulfate, by Merck Group (1 mentions)
Sn glycerol 3 phosphate, by Merck Group (1 mentions)
Succinate, by Merck Group (1 mentions)
Glutamate, by Merck Group (1 mentions)
Dimethyl fumarate, by Merck Group (1 mentions)
Ubiquinone, by Merck Group (1 mentions)
5 protocols using thenoyltrifluoroacetone
1
Investigating Autophagy Signaling Proteins
2
Mitochondrial Function Assays
3
Proteomic Analysis of Mitochondrial Dysfunction
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PARP antibody (Cell Signaling Technology), caspase 3 antibody (Cell Signaling Technology), GAPDH antibody (Santa Cruz Biotechnology), Anti‐Ubiquitin antibody (Sigma‐Aldrich), Anti‐ATP synthase antibody (merckmillipore), OPA1 antibody (Cell Signaling Technology), α‐Tubulin (Sigma‐Aldrich), CTR1 (PROTEINTECH), Pro‐Apoptosis Bcl‐2 Family Antibody Sampler Kit (Cell Signaling Technology) were used. All the inhibitors in this study were used at the indicated concentration unless stated independently: mitochondrial complex III inhibitor antimycin A (sigma A8674; 5 × 10−6m ), mitochondrial complex II inhibitor thenoyltrifluoroacetone (sigma T27006; 2 × 10−6m ), mitochondrial complex V inhibitor oligomycin (sigma 75351; 1 × 10−6m ), mitochondrial complex I inhibitor rotenone (sigma R8875; 5 × 10−9m ), cisplatin (sigma; 20 × 10−6m ), N‐acetyl‐L‐cysteine (sigma A7250; 5 × 10−3m ), MG132 (selleckchem S2619; 150 × 10−9m ), bortezomib (selleckchem S1013; 25 × 10−9m ), carfilzomib (selleckchem PR‐171; 10 × 10−9m ), FCCCP (sigma; 10 × 10−6m ). Corning matrigel matrix bulk (Corning), epidermal growth factor (Thermo), B27 supplement (Gibco), advanced DMEM/F12 (Gibco) were used. The methoxy PEG5000‐ PLGA (MW: 5000, mPEG‐PLGA) polymers were purchased from Xi'an Ruixi Biological Technology Co., Ltd., China.
4
SDS-PAGE Protein Analysis Reagents
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Sodium dodecyl sulfate (SDS), bovine serum albumin (BSA) and phenylmethane sulfonyl fluoride (PMSF) were purchased from Amresco (Solon, OH, USA). Phosphate-buffered saline (PBS), RIPA Lysis Buffer, SDS-PAGE kit and SDS loading buffer were obtained from Beyotime (Shanghai, China). Oligomycin A was purchased from Selleckchem (Houston, TX, USA). SNX482 was obtained from Alomone Labs (Jerusalem, Israel). Agarose, ethylenediaminetetraacetic acid (EDTA), methanol, paraformaldehyde, Tween 20, thenoyltrifluoroacetone (TTFA), polyformaldehyde, phosphatase inhibitor cocktail 3, dimethyl sulfoxide (DMSO), 4′,6-diamidino-2-phenylindole (DAPI) and N-formyl-Met-Leu-Phe (fMLP) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fluo-4/AM, Ca2+-free HBSS, Ca2+-containing HBSS, digitonin and Coomassie blue G-250 were obtained from Invitrogen (Carlsbad, CA, USA). Unless otherwise stated, all concentrations shown are the final concentrations.
5
Mitochondrial Function in Cardiac Cells
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Cardiac clusters were dissociated into single cells and stained with either JC-1 dye (10µg/mL; Thermo Fisher Scientific) or TMRM (0.25µM; Thermo Fisher Scientific) for 15 minutes at 37 o C. For mitochondrial complex inhibition studies, JC-1 stained cells were treated with rotenone, thenoyltrifluoroacetone and antimycin A (Sigma-Aldrich), respectively. Changes in JC-1 fluorescence intensity were measured using SpectraMax M3 (Molecular Devices, CA, USA). TMRM-stained cells were analyzed on BD FACSAria II (BD Biosciences, CA, USA). A total of 10,000 gated events were evaluated for each time-point and data analysis was performed using FlowJo software. Hexokinase Colorimetric Assay Kit (Sigma-Aldrich), L-Carnitine Assay Kit (Sigma-Aldrich) and ATP Determination Kit (Thermo Fisher Scientific) were used in this study. Cardiac clusters were lysed in respective buffers and assayed as per manufacturer's instructions.
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