Journal: Molecular Genetics & Genomic Medicine

Article Title: Biochemical phenotype of a common disease-causing mutation and a possible therapeutic approach for the phosphomannomutase 2-associated disorder of glycosylation

doi: 10.1002/mgg3.3

Figure Lengend Snippet: Ligand binding can affect the thermal stability of PMM2. Heat-induced melting profile of wild-type PMM2 (A and C) and F119L-PMM2 (B and D) were recorded by thermal shift assay and by circular dichroism. For thermal shift assay, the proteins (0.2 mg/mL) were equilibrated in buffer (HEPES 20 mmol/L, NaCl 150 mmol/L, pH 7.5) containing Sypro Orange2.5X and the appropriate ligands: MgCl 2 5 mmol/L, EDTA 5 mmol/L, Glc-6-P 0.5 mmol/L + MgCl 2 5 mmol/L, Glc-6-P 0.5 mmol/L + MgCl 2 5 mmol/L + vanadate 0.5 mmol/L, Glu-1,6-P 0.5 mmol/L + MgCl 2 5 mmol/L. The samples were distributed in 96-well PCR plates, the plates were sealed, and heated from 25 to 80° at 0.5°C/min. The experiment was run on an iCycler iQ Real Time PCR Detection System. An excitation wavelength of 490 nm and an emission wavelength of 575 nm were used to collect the data. When the melting profile was obtained by circular dichroism the proteins (0.2 mg/mL) were equilibrated in the same buffer in the presence of MgCl 2 1 mmol/L or EDTA 5 mmol/L. The signal at 222 nm was recorded while temperature was increased at 0.5°C/min from 20°C. The raw data were corrected by taking into account the slopes of the pre- and post-transition baselines, then they were normalized.

Article Snippet: The proteins (0.2 mg/mL) were equilibrated in the presence of HEPES 20 mmol/L, NaCl 150 mmol/L, pH 7.5, Sypro Orange 2.5× (Invitrogen Molecular Probes, http://lifetechnologies.com ).

Techniques: Ligand Binding Assay, Thermal Shift Assay, Gas Chromatography, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

Journal: Molecular Genetics & Genomic Medicine

Article Title: Biochemical phenotype of a common disease-causing mutation and a possible therapeutic approach for the phosphomannomutase 2-associated disorder of glycosylation

doi: 10.1002/mgg3.3

Figure Lengend Snippet: Specific activity of PMM2 depends on glucose-1,6-bisphosphate concentration. The assay was performed at 32°C in a reaction mixture containing HEPES 20 mmol/L, pH 7.5, MgCl 2 5 mmol/L, NaCl 150 mmol/L, NADP+ 0.25 mmol/L, Glu-1-P (0.04 or 0.60 mmol/L), and yeast glucose 6-phosphate dehydrogenase 10 μg/mL, while Glc-1,6-P 2 was changed in the range 0–80 μmol/L. Enzymes concentrations were 107 nmol/L for wt-PMM2 and 73 nmol/L for F119L-PMM2. The hyperbolic dependence of velocity on the activator concentration was fitted using Michaelis and Menten equation to evaluate EC50.

Article Snippet: The proteins (0.2 mg/mL) were equilibrated in the presence of HEPES 20 mmol/L, NaCl 150 mmol/L, pH 7.5, Sypro Orange 2.5× (Invitrogen Molecular Probes, http://lifetechnologies.com ).

Techniques: Activity Assay, Concentration Assay, Gas Chromatography

Journal: Molecular Genetics & Genomic Medicine

Article Title: Biochemical phenotype of a common disease-causing mutation and a possible therapeutic approach for the phosphomannomutase 2-associated disorder of glycosylation

doi: 10.1002/mgg3.3

Figure Lengend Snippet: Long-term stability of F119L-PMM2. F119L-PMM2 (0.027 mmol/L of monomer equivalents) was equilibrated in HEPES 50 mmol/L pH 7.1 containing NaCl 150 mmol/L. Aliquots containing 1.6 μg of protein were taken at known incubation time and diluted immediately to assay the residual activity with Glc-1-P under standard conditions. (A) Results obtained at 37°C in the presence of EDTA 0.1 mmol/L or MgCl 2 5 mmol/L. (B) Results obtained at 44°C in the presence of MgCl 2 5 mmol/L, MgCl 2 5 mmol/L plus Glc-1-P 0.5 mmol/L, MgCl 2 5 mmol/L plus Glc-1-P 0.5 mmol/L and vanadate 0.5 mmol/L or MgCl 2 5 mmol/L plus Glu-1,6-P 2 0.5 mmol/L

Article Snippet: The proteins (0.2 mg/mL) were equilibrated in the presence of HEPES 20 mmol/L, NaCl 150 mmol/L, pH 7.5, Sypro Orange 2.5× (Invitrogen Molecular Probes, http://lifetechnologies.com ).

Techniques: Incubation, Activity Assay, Gas Chromatography

Journal: Molecular Genetics & Genomic Medicine

Article Title: Biochemical phenotype of a common disease-causing mutation and a possible therapeutic approach for the phosphomannomutase 2-associated disorder of glycosylation

doi: 10.1002/mgg3.3

Figure Lengend Snippet: Ligand binding can increase the resistance to proteases of PMM2. Purified wild-type PMM2 and F119L-PMM2 (A) were incubated (0.5 mg/mL) with thermolysin in HEPES 20 mmol/L, NaCl 150 mmol/L, MgCl 2 0.1 mmol/L, pH 7.5 at the indicated protease substrate ratio (w/w) for 1 or 2 h at 37°C before they were analyzed (5 μg of each sample) by SDS-PAGE. Purified F119L-PMM2 (B) was incubated (0.2 mg/mL) with thermolysin in HEPES 20 mmol/L, NaCl 150 mmol/L, MgCl 2 0.1 mmol/L, pH 7.5 at the indicated protease substrate ratio (w/w), in the presence of no ligands, Glc-1,6-P 2 0.5 mmol/L or Glu-6-P 0.5 mmol/L plus vanadate 0.5 mmol/L, for 2 h at 37°C before they were analyzed (2 μg of each sample) by SDS-PAGE. The protein bands were visualized by Coomassie blue staining and the intensity of the bands quantified. The not-digested protein was quantified and expressed as percentage of the starting material (no protease panel A; time 0 panel B).

Article Snippet: The proteins (0.2 mg/mL) were equilibrated in the presence of HEPES 20 mmol/L, NaCl 150 mmol/L, pH 7.5, Sypro Orange 2.5× (Invitrogen Molecular Probes, http://lifetechnologies.com ).

Techniques: Ligand Binding Assay, Purification, Incubation, SDS Page, Gas Chromatography, Staining

Journal: Molecular Genetics & Genomic Medicine

Article Title: Biochemical phenotype of a common disease-causing mutation and a possible therapeutic approach for the phosphomannomutase 2-associated disorder of glycosylation

doi: 10.1002/mgg3.3

Figure Lengend Snippet: Ligand binding affects the quaternary structure of PMM2. Wild-type PMM2 (0.010 mg) and F119L-PMM2 (0.0065 mg) were subjected to size exclusion chromatography on BioSep-SEC-S3000 column equilibrated in HEPES 20 mmol/L pH 7.5, NaCl 150 mmol/L, MgCl 2 5 mmol/L (long dashed line for the wild-type PMM2 or short dashed line for F119L-PMM2) or in the same buffer containing Glc-6-P 0.5 mmol/L and vanadate 0.1 mmol/L (continuous line for wild-type PMM2 or dotted line for F119L-PMM2). The chromatography was run at room temperature at 0.5 mL/min.

Article Snippet: The proteins (0.2 mg/mL) were equilibrated in the presence of HEPES 20 mmol/L, NaCl 150 mmol/L, pH 7.5, Sypro Orange 2.5× (Invitrogen Molecular Probes, http://lifetechnologies.com ).

Techniques: Ligand Binding Assay, Size-exclusion Chromatography, Gas Chromatography, Chromatography

Journal: Molecular Genetics & Genomic Medicine

Article Title: Biochemical phenotype of a common disease-causing mutation and a possible therapeutic approach for the phosphomannomutase 2-associated disorder of glycosylation

doi: 10.1002/mgg3.3

Figure Lengend Snippet: Specific activity of F119L-PMM2 depends on enzyme concentration. The assay was performed at 32°C in a reaction mixture containing HEPES 20 mmol/L, pH 7.5, MgCl 2 5 mmol/L, NaCl 150 mmol/L, NADP+ 0.25 mmol/L, Glc-1,6-P 2 0.030 mmol/L, and yeast glucose 6-phosphate dehydrogenase 10 μg/mL. The reaction mixture also contained BSA at 0.5 mg/mL. Three sets of experiments were carried out in the presence of 0.04, 0.16, or 0.6 mmol/L Glc-1-P and the F119L-PMM2 concentration changed in the range 10–240 nmol/L (monomer equivalents).

Article Snippet: The proteins (0.2 mg/mL) were equilibrated in the presence of HEPES 20 mmol/L, NaCl 150 mmol/L, pH 7.5, Sypro Orange 2.5× (Invitrogen Molecular Probes, http://lifetechnologies.com ).

Techniques: Activity Assay, Concentration Assay, Gas Chromatography

Journal: Molecular Genetics & Genomic Medicine

Article Title: Biochemical phenotype of a common disease-causing mutation and a possible therapeutic approach for the phosphomannomutase 2-associated disorder of glycosylation

doi: 10.1002/mgg3.3

Figure Lengend Snippet: Specific activity of wt-PMM2 changes as a function of protein concentration. The assay was performed at 32°C in a reaction mixture containing HEPES 20 mmol/L, pH 7.5, MgCl 2 5 mmol/L, NaCl 150 mmol/L, NADP+ 0.25 mmol/L, Glc-1,6-P 2 0.030 mmol/L and yeast glucose 6-phosphate dehydrogenase 0.010 mg/mL. The reaction mixture also contained Glc-1-P 0.020 mmol/L and BSA 0.5 mg/mL. The wt-PMM2 concentration changed in the range 2–110 nmol/L (monomer equivalents).

Article Snippet: The proteins (0.2 mg/mL) were equilibrated in the presence of HEPES 20 mmol/L, NaCl 150 mmol/L, pH 7.5, Sypro Orange 2.5× (Invitrogen Molecular Probes, http://lifetechnologies.com ).

Techniques: Activity Assay, Protein Concentration, Gas Chromatography, Concentration Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Perlecan is required to inhibit thrombosis after deep vascular injury and contributes to endothelial cell-mediated inhibition of intimal hyperplasia

doi:

Figure Lengend Snippet: Expression of perlecan antisense leads to reduced perlecan secretion and loss of FGF-2 binding inhibition in endothelial cell-conditioned media. ( A ) Perlecan antisense construct. ( B ) Western blot of conditioned media from untransfected BAE cells, and clones transfected with the antiperlecan sequences (BAE-AP1–6) or the vector alone (BAE-NEO1–4). All samples were normalized for cell number (1 × 10 4 cells) and subjected to heparinase treatment before gel electrophoresis. ( C ) FGF-2 binding to smooth muscle cells is inhibited by conditioned media from BAE-NEO cells to a greater extent than by that from BAE-AP cells 125 ). VSMCs were plated (5 × 10 4 cells/well) in 24-well culture plates (Costar) in DMEM, 10% calf serum. When the cells reached confluence (day 3), the medium was removed, and the monolayers were washed with cold (0°C) binding buffer (DMEM/25 mM Hepes/0.05% gelatin), and binding of 125 I-FGF-2 was conducted in the presence of conditioned media (equivalent to the media conditioned by 3.5 × 10 4 cells) for 2.5 h at 4°C. The data shown are the averages ± SEM of triplicate determinations. Control binding represented the amount of 125 I-FGF-2 bound to cells in the absence of any conditioned media (100%).

Article Snippet: Heparinase III, Chondroitinase ABC, heparan sulfate from bovine kidney, gelatin, Hepes, and trypsin were from Sigma.

Techniques: Expressing, Binding Assay, Inhibition, Construct, Western Blot, Clone Assay, Transfection, Plasmid Preparation, Nucleic Acid Electrophoresis

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Functional and Morphological Analysis of OFF Bipolar Cells

doi: 10.1007/978-1-4939-7720-8_15

Figure Lengend Snippet: Preparation for the eye dissection. (A) A ventilated cart to place the animal cage inside for dark adaptation. (B) A handmade chopper with a razor blade (red arrow) inserted. A micrometer dial on the bottom moves the slicing chamber laterally during the dissection. (C) Grease pens are assembled from a 1 ml syringe and a pipetter tip (~200 µl). Vacuum grease is inserted into the back of the syringe. (D) A dark box for holding retinal preparations. The bottom is filled with water and oxygen is bubbled continuously. When the box is closed, the inside is completely dark and a moist, oxygenated environment is continually provided. Preparations sit on the shelf. (E) A dissecting chamber filled with HEPES solution. PE tubing from an oxygen source continuously delivers oxygen to the retinal tissue. (F) All tools and pipettes are arranged next to the dissecting microscope, with HEPES solution on ice nearby. (G) A slicing chamber to hold retina tissue (white arrow) attached to the Millipore filter paper (green arrow). (H) A plastic coverslip with parallel grease rails (yellow asterisks) and an attached retinal slice (white arrow) with filter paper (green arrow) set into the grease. (I) A micro-pipette filler and a 1 ml pipette for reference.

Article Snippet: Immediately after the solution is removed, a halved piece of Millipore filter paper is placed on top of the retinal slab and a drop of HEPES solution is placed on top of the Millipore filter paper.

Techniques: Dissection, Microscopy, Transferring

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: pH-sensitive niosomes: Effects on cytotoxicity and on inflammation and pain in murine models

doi: 10.1080/14756366.2016.1268607

Figure Lengend Snippet: Release profiles of IBU and LID from the vesicular carriers in HEPES (pH =7.4) at 32 °C as a function of time: (A) TW20 samples; (B) TW2–0GLY samples. Release experiments were carried out in triplicate. The reported value represents mean values and lay within 10% of the mean.

Article Snippet: Materials Tween-20 (Tw20), Sephadex G-75, Hepes salt [N -(2-hydroxyethyl) piperazine-N -(2-ethanesulfonic acid)], calcein, Ibuprofen and Lidocaine were purchased from Sigma-Aldrich (Sigma-Aldrich SRL, Milan, Italy).

Techniques:

Journal: Molecular Biology of the Cell

Article Title: Endosome to Golgi Transport of Ricin Is Regulated by Cholesterol

doi:

Figure Lengend Snippet: Effect of mβCD and mβCD/chol on sulfation of ricin sulf-2. (A) Representative example of the resulting bands seen after fluorography in a given experiment. The HeLa-TetOn/Rab9S21N cells were washed with sulfate-free DME medium and incubated for 4 h in the presence of Na 2 35 SO 4 before addition of 5 mM mβCD or mβCD/chol. After 30 min, ricin sulf-2 was added, and the incubation continued for 2 h. The cells were then washed with 0.1 M lactose in HEPES medium at 37°C and with ice-cold PBS before they were lysed. The nuclei were removed by centrifugation, and the sulfated ricin was immunoprecipitated with rabbit antiricin antibodies attached to protein A-sepharose overnight at 4°C. The immunoprecipitate was analyzed by SDS-PAGE (12%) under reducing conditions followed by fluorography. The intensities of the resulting bands were determined by densiometric quantitation using ImageQuant 5.0. (B) Averaged data of three independent experiments.

Article Snippet: The cells were washed twice with HEPES medium and preincubated with 5 mM mβCD or mβCD/chol for 30 min at 37°C before addition of a monovalent conjugate of ricin B-chain coupled covalently to HRP (Sigma type IV).

Techniques: Incubation, Centrifugation, Immunoprecipitation, SDS Page, Quantitation Assay

Journal: Molecular Biology of the Cell

Article Title: Endosome to Golgi Transport of Ricin Is Regulated by Cholesterol

doi:

Figure Lengend Snippet: The effect of mβCD and mβCD/chol on the cytotoxicity of ricin in the HeLa-TetOn/Rab9S21N cell line. (A) Representative example of the toxic effect of increasing concentrations of ricin in a given experiment. (B) Averaged data of four independent experiments. The cells were washed twice with HEPES medium lacking leucine and incubated with or without 5 mM mβCD and mβCD/chol for 30 min at 37°C before increasing concentrations of ricin were added. After incubating the cells for 2 h, the toxic effect was measured as described in Material and Methods.

Article Snippet: The cells were washed twice with HEPES medium and preincubated with 5 mM mβCD or mβCD/chol for 30 min at 37°C before addition of a monovalent conjugate of ricin B-chain coupled covalently to HRP (Sigma type IV).

Techniques: Incubation

Journal: Molecular Biology of the Cell

Article Title: Endosome to Golgi Transport of Ricin Is Regulated by Cholesterol

doi:

Figure Lengend Snippet: Effect of mβCD and mβCD/chol on the recycling (A) and degradation (B) of ricin in the HeLa-TetOn/Rab9S21N cell line. The cells were washed twice with HEPES medium, and incubated with or without 5 mM mβCD and mβCD/chol for 30 min at 37°C. 125 I-labeled ricin was then added, and the cells incubated further for 20 min before the amount of recycled and degraded ricin was measured as described in Material and Methods. The error bars show deviations between duplicates of a typical experiment.

Article Snippet: The cells were washed twice with HEPES medium and preincubated with 5 mM mβCD or mβCD/chol for 30 min at 37°C before addition of a monovalent conjugate of ricin B-chain coupled covalently to HRP (Sigma type IV).

Techniques: Incubation, Labeling

Journal: Molecular Biology of the Cell

Article Title: Endosome to Golgi Transport of Ricin Is Regulated by Cholesterol

doi:

Figure Lengend Snippet: Effect of mβCD and mβCD/chol on endocytosis of transferrin (A) and ricin in the absence (B) and presence of monensin (5 μM) (C). The HeLa-TetOn/Rab9S21N cells were washed twice in HEPES medium and incubated with or without 5 mM mβCD and mβCD/chol for 30 min at 37°C before 125 I-labeled transferrin or 125 I-labeled ricin were added to the cells. The amounts of endocytosed transferrin was measured after 5 min, while the amounts of endocytosed ricin was measured after 15 min and 2 h as described in Material and Methods. The error bars show deviations between duplicates of a typical experiment.

Article Snippet: The cells were washed twice with HEPES medium and preincubated with 5 mM mβCD or mβCD/chol for 30 min at 37°C before addition of a monovalent conjugate of ricin B-chain coupled covalently to HRP (Sigma type IV).

Techniques: Incubation, Labeling

Journal: Macromolecular rapid communications

Article Title: Ultrasensitive Detection of Proteins on Western Blots with Semiconducting Polymer Dots

doi: 10.1002/marc.201200809

Figure Lengend Snippet: (A) Structure of CN-PPV polymer and the absorption (dashed) and emission (solid) spectra of CN-PPV Pdot (black) and CN-PPV Pdot-streptavidin conjugate (CNPPV-Strep, red) in a 0.1% PEG 20 mM HEPES buffer. (B) The absorption and emission spectra of Qdot

Article Snippet: To conjugate Pdots with streptavidin, to this 5 mL of Pdot solution, we added 100 μL of PEG (5 % w/v in water, MW 3350), 100 μL of 1 M HEPES buffer (pH 7.4), 300 μL of streptavidin (1 mg/mL in 20 mM HEPES buffer), and 50 μL of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC, from Sigma-Aldrich) (10 mg/mL in water).

Techniques:

Journal: PLoS ONE

Article Title: Glutamate Uptake Triggers Transporter-Mediated GABA Release from Astrocytes

doi: 10.1371/journal.pone.0007153

Figure Lengend Snippet: Glu-induced GABA release is not mediated by vesicular release. (A) Elevation of [GABA] o in acute rat hippocampal slices in control conditions (n = 6) and in the presence of glutamate receptor antagonists (n = 7; P = 0.014; 128.5±13.4% of pre-stimulus control). (B) Cytosolic GABA level as determined by [ 3 H]GABA uptake in the presence of 100 µM NNC-711 and Glu receptor antagonists in rat cerebrocortical NPMV fractions (n = 4; P = 0.82) (C) Elevation of [GABA] o in acute rat hippocampal slices in control conditions (n = 6) and in low-[Ca 2+ ]/high-[Mg 2+ ] buffer (n = 5; P = 0.91; 148.3±15.5% of pre-stimulus control). (D) Cytosolic GABA level as determined by [ 3 H]GABA uptake in the presence of 100 µM NNC-711 in low-[Ca 2+ ] buffer in rat cerebrocortical NPMV fractions (n = 3; P = 0.49). Low-[Ca 2+ ] buffer contained 145 mM NaCl, 5 mM KCl, 20 mM MgCl 2 , 10 mM glucose and 20 mM HEPES (pH 7.5).

Article Snippet: Uptake studies were performed as follows: aliquots of NPMV fractions in HEPES buffer were preincubated with test compounds in the presence of NNC-711 (100 µM, Tocris) at 30°C for 10 min.

Techniques:

Journal: Inflammation

Article Title: Off-Target Anti-Inflammatory Activity of the P2X7 Receptor Antagonist AZ11645373

doi: 10.1007/s10753-016-0499-8

Figure Lengend Snippet: AZ11645373 inhibits OxPAPC-induced upregulation of IL-8 ( a ) and COX-2 ( b ) mRNA in endothelial cells. HUVECtert cells were preincubated with AZ11645373 in EBM-2 medium containing 2% FCS and 20 mM HEPES at 37 °C. After 20 min, OxPAPC was added and the cells were further incubated for 7 h. The isolation of total RNA, cDNA synthesis, and real-time PCR were performed as described in the “ Materials and Methods ” section. Results are normalized to β2-microglobulin. * p

Article Snippet: Stimulation of cells was performed in endothelial basal medium-2 (EBM-2) (Lonza) supplemented with 2% FCS (Sigma-Aldrich), and 20 mM HEPES (Lonza).

Techniques: Incubation, Isolation, Real-time Polymerase Chain Reaction

Journal: Inflammation

Article Title: Off-Target Anti-Inflammatory Activity of the P2X7 Receptor Antagonist AZ11645373

doi: 10.1007/s10753-016-0499-8

Figure Lengend Snippet: AZ11645373 inhibits OxPAPC-induced IL-8 release in endothelial cells. a OxPAPC induces IL-8 release. HUVECtert cells were stimulated by OxPAPC at the indicated concentrations for 6 h in EBM-2/2% FCS/20 mM HEPES in a 96-well plate. The concentrations of IL-8 released into the culture medium were measured by ELISA. IL-8 produced by mock-treated cells (26 ± 1.1 pg/ml) was taken as 100%. *** p

Article Snippet: Stimulation of cells was performed in endothelial basal medium-2 (EBM-2) (Lonza) supplemented with 2% FCS (Sigma-Aldrich), and 20 mM HEPES (Lonza).

Techniques: Enzyme-linked Immunosorbent Assay, Produced

Journal: Nature Communications

Article Title: Active and dynamic mitochondrial S-depalmitoylation revealed by targeted fluorescent probes

doi: 10.1038/s41467-017-02655-1

Figure Lengend Snippet: In vitro activity of mitoDPP-3. a Structure of mitoDPP-3. b In vitro fluorescence assay of 1 µM mitoDPP-3 in HEPES (20 mM, pH 7.4, 150 mM NaCl, 0.1% Triton X-100) with either 50 nM purified APT1 or APT2 (λ ex 490/20 nm, λ em 545/20 nm). For plots, n = 3, error bars are ± s.e.m. c Fluorescence emission spectra at 30 min from probes as treated in ( b ). d UV–vis spectra of 25 µM mitoDPP-3 (black; normalized at 275 nm) and deprotected fluorophore product (gray; normalized at 513 nm) in HEPES (20 mM, pH 7.4, 150 mM NaCl, 0.1% Triton X-100). MitoDPP-3 shows UV–vis absorbance at 300 nm with extinction coefficient 12.6 × 10 3 M −1 cm −1

Article Snippet: In vitro kinetic assays of mitoDPP-2 and mitoDPP-3 In total 100 µL of 3 µM mitoDPP-2 or mitoDPP-3 in HEPES buffer (20 mM, pH = 7.4, 150 mM NaCl, 0.1% Triton X-100) were added to a 96-well optical bottom plate (Nunc 265301, Thermo Scientific) at room temperature.

Techniques: In Vitro, Activity Assay, Fluorescence, Purification

Journal: Nature Communications

Article Title: Active and dynamic mitochondrial S-depalmitoylation revealed by targeted fluorescent probes

doi: 10.1038/s41467-017-02655-1

Figure Lengend Snippet: Synthesis and in vitro activity of mitoDPP-2. a Synthetic scheme for mitoDPP-2. b In vitro fluorescence assay of mitoDPP-2 (1 µM) in HEPES (20 mM, pH 7.4, 150 mM NaCl, 0.1% Triton X-100) with either 50 nM purified APT1 or APT2 (λ ex 490/20 nm, λ em 545/20 nm). Error bars are ± s.e.m. ( n = 3). c Fluorescence emission spectra at 30 min from probes as treated in ( b ). d UV–vis spectra of 25 µM mitoDPP-2 (black; normalized at 275 nm) and deprotected fluorophore product (gray; normalized at 513 nm) in HEPES (20 mM, pH 7.4, 150 mM NaCl, 0.1% Triton X-100). MitoDPP-2 shows UV–vis absorbance at 300 nm with extinction coefficient 8.7 × 10 3 M −1 cm −1 . The deprotected fluorophore product shows a major UV-Vis absorbance peak at 513 nm with extinction coefficient 11.8 × 10 3 M −1 cm −1

Article Snippet: In vitro kinetic assays of mitoDPP-2 and mitoDPP-3 In total 100 µL of 3 µM mitoDPP-2 or mitoDPP-3 in HEPES buffer (20 mM, pH = 7.4, 150 mM NaCl, 0.1% Triton X-100) were added to a 96-well optical bottom plate (Nunc 265301, Thermo Scientific) at room temperature.

Techniques: In Vitro, Activity Assay, Fluorescence, Purification