recombinant human cox2  (R&D Systems)

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The Trypanosome-Derived Metabolite Indole-3-Pyruvate Inhibits Prostaglandin Production in Macrophages by Targeting COX2.

doi: 10.4049/jimmunol.2100402

Figure Lengend Snippet: FIGURE 2. I3P boosts COX2 expression. (A) BMDMs were treated with 1 mM I3P for 1 h prior to stimulation with LPS (100 ng/ml) for 8 h. The cells were lysed, mRNA was extracted, and the expression of Ptgs2 was quantified by qPCR (n 5 3 from three independent experiments). (B) BMDMs were treated with 1 mM I3P for 1 h prior to stimulation with LPS (100 ng/ml) for 6 and 24 h. (C) COX2 was measured by Western blotting (n 5 4 from three independent experiments), and quantification by densitometry is shown. (D) BMDMs were treated with various concentrations (31.25 mM to 1000 mM) of I3P or control media for 1 h prior to stimulation with LPS (100 ng/ml) for 24 h. (E) COX2 was measured by Western blotting (n 5 5 from three independent experiments), and quantification by densitometry is shown. (FH) BMDMs were treated with 1 mM I3P for 1 h prior to stimulation with LPS (100 ng/ml) for 4, 6, or 8 h. The expression of Ptges (F), Pgis (G), and Tbxas1 (H) was measured by qPCR (n 5 3 from three independent experiments). All genes were normalized to rps18. (I) BMDMs were treated with 1 mM I3P for 1 h prior to stimulation with LPS (100 ng/ml) for 24 h. The concentration of PGs in both the cell lysates and supernatants was quantified by ELISA (n 5 2 from two independent experiments). Densitometry analysis is presented as mean ± SD. All other data are mean ± SEM. A one-way ANOVA was performed for (A), (C), and (E). A two-way ANOVA was performed for (F) and (G). The data show the adjusted p value obtained from multiple comparisons, corrected for using the Tukey test for one-way ANOVA or the Sidak test for two-way ANOVA. **p < 0.005, ***p < 0.0005, ****p < 0.0001.

Article Snippet: A total of 1 mg recombinant human COX2 (R&D Systems) was used per well, and for this experiment, DMSO was used as the solvent for I3P rather than cell culture media.

Techniques: Expressing, Western Blot, Control, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The Trypanosome-Derived Metabolite Indole-3-Pyruvate Inhibits Prostaglandin Production in Macrophages by Targeting COX2.

doi: 10.4049/jimmunol.2100402

Figure Lengend Snippet: FIGURE 3. I3P inhibits COX2 activity. (A) Schematic showing the structures of indole-3-acetic acid, indomethacin, and I3P. (B) BMDMs were treated with indomethacin (50 or 100 mM), I3P (0.5 or 1 mM), or the relevant vehicle control (DMSO or media) for 1 h prior to stimulation with LPS (100 ng/ml) for 24 h, and COX2 levels were measured by Western blotting (n 5 3 from three independent experiments). (C) Quantification by densitometry is shown. (D) Schematic depicting hypothesis of I3P inhibition of COX2. (E) BMDMs were treated with 1 mM I3P for 45 min, followed by addition of 5 mM AA or vehicle (DMSO) for a further 15 min, prior to stimulation with LPS (100 ng/ml) for 24 h. The concentration of PGs in the supernatants was quantified by ELISA (E) (n 5 5 from three independent experiments). (F) COX2 activity was measured after incubation of 1 mg recombinant COX2 with various concen- trations (31.25 mM to 1000 mM) of I3P or vehicle control (DMSO) (n 5 3 from three independent experiments). Densitometry analysis is presented as mean ± SD. All other data are mean ± SEM. A one-way ANOVA was performed for (C). A two-way ANOVA was performed for (E). The data show the adjusted p value obtained from multiple comparisons, corrected for using the Tukey test for one-way ANOVA or the Sidak test for two-way ANOVA. *p < 0.05, ****p < 0.0001.

Article Snippet: A total of 1 mg recombinant human COX2 (R&D Systems) was used per well, and for this experiment, DMSO was used as the solvent for I3P rather than cell culture media.

Techniques: Activity Assay, Control, Western Blot, Inhibition, Concentration Assay, Enzyme-linked Immunosorbent Assay, Incubation, Recombinant

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The Trypanosome-Derived Metabolite Indole-3-Pyruvate Inhibits Prostaglandin Production in Macrophages by Targeting COX2.

doi: 10.4049/jimmunol.2100402

Figure Lengend Snippet: FIGURE 4. The augmentation of COX2 expression by I3P is regulated by a PG feedback loop. (A) BMDMs were treated with 10 mM PGE2 for 1 h and subsequently with 1 mM I3P for 1 h further, prior to stimulation with LPS (100 ng/ml) for 24 h. (B) COX2 was measured by Western blotting (n 5 3 from three inde- pendent experiments), and quantification by densitometry is shown. (C) BMDMs were treated with 5 mM of the EP2 antago- nist AH6809 for 1 h and subsequently with 1 mM I3P for 1 h fur- ther, prior to stimulation with LPS (100 ng/ml) for 24 h. (D) COX2 was measured by Western blotting (n 5 4 from three inde- pendent experiments), and quantification by densitometry is shown. (E) BMDMs were treated with 10 mM of the EP4 antago- nist GW 627368X for 30 min and subsequently with 0.5 mM I3P for 1 h further, prior to stimulation with LPS (100 ng/ml) for 24 h. (F) COX2 was measured by Western blotting (n 5 4 from three independent experiments), and quantification by densitome- try is shown. (G) BMDMs were treated with 10 mM forskolin for 30 min and subsequently with 1 mM I3P for 1 h further, prior to stimulation with LPS (100 ng/ml) for 24 h. (H) COX2 was mea- sured by Western blotting (n 5 4 from three independent experi- ments), and quantification by densitometry is shown. Densitometry analysis is presented as mean ± SD. A one-way ANOVA was per- formed. The data show the adjusted p value obtained from multiple comparisons, corrected for using the Tukey test. *p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.0001.

Article Snippet: A total of 1 mg recombinant human COX2 (R&D Systems) was used per well, and for this experiment, DMSO was used as the solvent for I3P rather than cell culture media.

Techniques: Expressing, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The Trypanosome-Derived Metabolite Indole-3-Pyruvate Inhibits Prostaglandin Production in Macrophages by Targeting COX2.

doi: 10.4049/jimmunol.2100402

Figure Lengend Snippet: FIGURE 5. The augmentation of COX2 expression by I3P is partially dependent on AhR activation. (A) BMDMs from AhR1/1 and AhR/ mice were pretreated with 10 mM 3MC for 30 min prior to stimulation with LPS (100 ng/ml) for 24 h. (B) COX2 levels were measured by Western blotting (n 5 3 from one independent experiment), and quantification by densitometry is shown. (C) BMDMs from AhR1/1 and AhR/

Article Snippet: A total of 1 mg recombinant human COX2 (R&D Systems) was used per well, and for this experiment, DMSO was used as the solvent for I3P rather than cell culture media.

Techniques: Expressing, Activation Assay, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The Trypanosome-Derived Metabolite Indole-3-Pyruvate Inhibits Prostaglandin Production in Macrophages by Targeting COX2.

doi: 10.4049/jimmunol.2100402

Figure Lengend Snippet: FIGURE 6. I3P blocks trypanosome lysate-induced PG production and LPS-induced PG production in human macrophages. (AC) BMDMs were pre- treated with 1 mM I3P or control media for 30 min before they were treated with 100 ng/ml IFN-g and 25 or 100 mg/ml trypanosome lysate for 24 h. COX2 expression was measured by Western blotting (n 5 3 from three independent experiments) (A), and quantification by densitometry is shown (B). (C) PGs were measured by ELISA (n 5 3 from three independent experiments). (DF) Primary human macrophages were pretreated with I3P (1 mM, 0.5 mM, or 0.25 mM) or control media for 1 h before they were treated with LPS (100 ng/ml) for 24 h. COX2 expression was measured by Western blotting (n 5 4 from three independent experiments) (D), and quantification by densitometry is shown (E). (F) PGs were measured by ELISA (n 5 4 from three independent experiments). (G) Schematic representation of inhibition of COX2 by I3P. Densitometry analysis is presented as mean ± SD. All other data are mean ± SEM. A one-way ANOVA was performed. The data show the adjusted p value obtained from multiple comparisons, corrected for using the Tukey test. ***p < 0.0005, ****p < 0.0001.

Article Snippet: A total of 1 mg recombinant human COX2 (R&D Systems) was used per well, and for this experiment, DMSO was used as the solvent for I3P rather than cell culture media.

Techniques: Control, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Inhibition

Journal: Molecules

Article Title: An Investigation into the Interaction between Double Hydroxide-Based Antioxidant Benzophenone Derivatives and Cyclooxygenase 2

doi: 10.3390/molecules26216622

Figure Lengend Snippet: Sensograms for the interaction of 12 compounds with cyclooxygenases 2 (COX2) obtained with biolayer interferometry (BLI); ( a ) All 12 compounds were initially assayed at a single concentration of 100 μM. ( b – d ) The results showed a medium-high affinity of compounds DB2 ( b ), SC2 ( c ) and YB2 ( d ) to COX2.

Article Snippet: Recombinant active human COX2 was purchased from Sino Biological Co. (Beijing, China).

Techniques: Concentration Assay

Journal: Molecules

Article Title: An Investigation into the Interaction between Double Hydroxide-Based Antioxidant Benzophenone Derivatives and Cyclooxygenase 2

doi: 10.3390/molecules26216622

Figure Lengend Snippet: 3D putative binding complexes and hydrogen bonding of compounds DB2 ( a , b ), SC2 ( c , d ) and YB2 ( e , f ) with the COX2 enzyme (PDB code 5f1a). (COX2 is shown as cyan ribbon; The ligand surface is indicated in purple; Compounds DB2, SC2 and YB2 are shown in capped sticks models; The amino acids involved in hydrogen bond contact are shown in line models; Hydrogen bonding are yellow lines).

Article Snippet: Recombinant active human COX2 was purchased from Sino Biological Co. (Beijing, China).

Techniques: Binding Assay

Journal: Molecules

Article Title: An Investigation into the Interaction between Double Hydroxide-Based Antioxidant Benzophenone Derivatives and Cyclooxygenase 2

doi: 10.3390/molecules26216622

Figure Lengend Snippet: Spectra of COX2 (6.0 × 10 −8 mol∙L −1 ) fluorescence quenching upon binding to compounds DB2 ( a , b ), SC2 ( c , d ) or YB2 ( e , f ) (1→7: 0, 0.75, 1.50, 2.25, 3.00, 3.75, 4.50 × 10 −5 mol∙L −1 ) at 298 K and 310 K.

Article Snippet: Recombinant active human COX2 was purchased from Sino Biological Co. (Beijing, China).

Techniques: Fluorescence, Binding Assay

Journal: Molecules

Article Title: An Investigation into the Interaction between Double Hydroxide-Based Antioxidant Benzophenone Derivatives and Cyclooxygenase 2

doi: 10.3390/molecules26216622

Figure Lengend Snippet: Stern-Volmer plots for COX2-DB2 ( a ), COX2-SC2 ( b ) and COX2-YB2 ( c ) at 298 K and 310 K; Double logarithmic plots for COX2-DB2 ( d ), COX2-SC2 ( e ) and COX2-YB2 ( f ) at 298 K and 310 K; The overlap between COX2 (5.0 × 10 −8 mol∙L −1 ) fluorescence spectra (solid line) and compounds DB2 ( g ), SC2 ( h ) or YB2 ( i ) (5.0 × 10 −8 mol∙L −1 ) absorbance spectra (short dot).

Article Snippet: Recombinant active human COX2 was purchased from Sino Biological Co. (Beijing, China).

Techniques: Fluorescence

Journal: Molecules

Article Title: An Investigation into the Interaction between Double Hydroxide-Based Antioxidant Benzophenone Derivatives and Cyclooxygenase 2

doi: 10.3390/molecules26216622

Figure Lengend Snippet: The Stern-Volmer quenching constant ( Ksv ), bimolecular quenching constant ( Kq ) and binding constant ( K A ) for the interaction of compounds DB2, SC2 and YB2 with COX2 at 298 K and 310 K.

Article Snippet: Recombinant active human COX2 was purchased from Sino Biological Co. (Beijing, China).

Techniques: Binding Assay, Control

Journal: Molecules

Article Title: An Investigation into the Interaction between Double Hydroxide-Based Antioxidant Benzophenone Derivatives and Cyclooxygenase 2

doi: 10.3390/molecules26216622

Figure Lengend Snippet: Thermodynamic parameters, the effective spectral overlap ( J ) and the separation distance ( r ) for the interaction of compounds DB2, SC2 and YB2 with COX2 at 298 K and 310 K.

Article Snippet: Recombinant active human COX2 was purchased from Sino Biological Co. (Beijing, China).

Techniques:

Journal: Molecules

Article Title: An Investigation into the Interaction between Double Hydroxide-Based Antioxidant Benzophenone Derivatives and Cyclooxygenase 2

doi: 10.3390/molecules26216622

Figure Lengend Snippet: Synchronous fluorescence spectra of COX2 (6.0 × 10 −8 mol∙L −1 ) in the presence of different concentrations of compounds DB2 ( a , b ), SC2 ( c , d ) and YB2 ( e , f ) (1→7: 0, 0.75, 1.50, 2.25, 3.00, 3.75, 4.50 × 10 −5 mol∙L −1 ) at 298 K.

Article Snippet: Recombinant active human COX2 was purchased from Sino Biological Co. (Beijing, China).

Techniques: Fluorescence

Journal: Molecules

Article Title: An Investigation into the Interaction between Double Hydroxide-Based Antioxidant Benzophenone Derivatives and Cyclooxygenase 2

doi: 10.3390/molecules26216622

Figure Lengend Snippet: Three-dimensional fluorescence contour spectra of COX2 (5.0 × 10 −8 mol∙L −1 ) in the absence ( a ) and presence of compounds DB2 ( b ), SC2 ( c ) or YB2 ( d ) (4.50 × 10 −5 mol∙L −1 ) at 298 K.

Article Snippet: Recombinant active human COX2 was purchased from Sino Biological Co. (Beijing, China).

Techniques: Fluorescence

Journal: Molecules

Article Title: An Investigation into the Interaction between Double Hydroxide-Based Antioxidant Benzophenone Derivatives and Cyclooxygenase 2

doi: 10.3390/molecules26216622

Figure Lengend Snippet: The characteristic parameters of three-dimensional fluorescence spectra.

Article Snippet: Recombinant active human COX2 was purchased from Sino Biological Co. (Beijing, China).

Techniques: Fluorescence