fluorogenic peptide substrate  (R&D Systems)

Journal: Advanced Science

Article Title: Membrane‐Active Peptide Protects Against Inflammation by Targeting NLRP3 Activation at the Trans‐Golgi Network

doi: 10.1002/advs.76587

Figure Lengend Snippet: Pre‐incubation with Pep19‐2.5 inhibits nigericin‐ and MSU crystal‐induced IL‐1β secretion. (A) Primary monocytes were primed with 1 µg/mL Pam 3 CSK 4 for 3 h and then stimulated with 10 µM nigericin or 200 µg/mL MSU crystals for 3 h. Pep19‐2.5 was added 30 min before priming at increasing concentrations ranging from 0.4 to 18 µM. Supernatants were analyzed for IL‐1β by ELISA. Stimulus‐induced IL‐1β secretion was set to 100%. Mean ± SEM ( n = 4 biologically independent experiments for nigericin, n = 2 biologically independent experiments for MSU crystals). One‐sample t ‐test against 100%. (B) hMDMs were primed with 1 µg/mL Pam 3 CSK 4 for 4 h and then stimulated with 5 µM nigericin for 2 h. Pep19‐2.5 was added at the indicated concentrations 30 min before priming. Supernatants were analyzed for IL‐1β by ELISA. Stimuli‐induced IL‐1β secretion was set to 100%. Mean + SEM ( n = 4 biologically independent experiments). One‐sample t ‐test against 100%. (C) THP‐1 macrophages were primed and stimulated as described in (A). For inhibition experiments, 18 µM Pep19‐2.5 was added 30 min before priming. Supernatants were analyzed for IL‐1β by ELISA. Stimulus‐induced IL‐1β secretion was set to 100%. Mean + SEM ( n = 4 biologically independent experiments for nigericin, n = 3 biologically independent experiments for MSU crystals). One‐sample t ‐test against 100%. (D and E) THP‐1 macrophages were primed and treated with NLRP1 (D) or AIM2 (E) activators. Supernatants were analyzed for IL‐1β by ELISA. Stimulus‐induced IL‐1β secretion was set to 100%. Mean + SEM ( n = 3 biologically independent experiments). One‐sample t ‐test against 100%. (F–H) Protein expression of pro‐IL‐1β (F), full‐length (G), and cleaved GSDMD (H) were analyzed by Western blot. Basal, Pam 3 CSK 4 ‐ or nigericin‐induced expression was set to 100%. Mean + SEM ( n = 3 biologically independent experiments). One‐sample t ‐test against 100%. (I) THP‐1 macrophages were primed and stimulated after peptide incubation as described in (A). After 1 h of stimulation with nigericin, the medium was removed, and Z‐WEHD buffer was added according to the manufacturer´s protocol. Ac‐YVAD‐CHO control was subtracted, and stimulus‐induced caspase‐1 activity was normalized to 100%. Mean ± SEM ( n = 3 biologically independent experiments). One‐sample t ‐test against 100%. (J) hMDMs from healthy donors were seeded in µ‐Slides VI and incubated at 37°C in a humidified atmosphere of 5% CO 2 for 1 h. Macrophages were primed with 1 µg/mL Pam 3 CSK 4 for 3 h and stimulated with 10 µM nigericin for 1 h in the presence of fluorophore‐conjugated Rh‐Pep19‐2.5. Active caspase‐1 was stained with the caspase‐1 pseudosubstrate FLICA660‐YVAD‐FMK. Cells were washed and fixed, and nuclei were stained with Hoechst 34580. Confocal microscopy images are representative of n = 2 biologically independent experiments. Scale bar is 10 µm (upper panel) and 2 µm (zoomed in lower panel). (K) Solutions containing 0.5 µM Atto488‐conjugated Pep19‐2.5 were prepared in the presence of increasing concentrations of recombinant human caspase‐1 and incubated overnight at 4°C under gentle agitation. Microfluidic diffusional sizing (MDS) measurements were performed at room temperature to determine the hydrodynamic radius ( R h ) of fluorescently labeled particles. Dots represent the mean ± SD of replicates ( n ≥ 3) of free peptide and peptide–protein complexes, and the black line represents the best nonlinear fit according to Equation . (L) Human recombinant caspase‐1 was incubated with the caspase‐1‐specific substrate Ac‐YVAD‐AMC at the indicated concentrations of Pep19‐2.5 or 40 µM of the caspase‐1 inhibitor VX765. Enzyme activity in relative fluorescence units (RFU) was measured every 2 min at 37°C. Representative traces are means ± SEM ( n = 2 biologically independent experiments).

Article Snippet: The fluorogenic caspase‐1 substrate Ac‐YVAD‐AMC (HY‐P2717, MedChemExpress, NJ, USA) was added to a final concentration of 10 μM.

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Inhibition, Expressing, Western Blot, Control, Activity Assay, Staining, Confocal Microscopy, Recombinant, Gentle, Labeling, Fluorescence

Journal: Advanced Science

Article Title: Membrane‐Active Peptide Protects Against Inflammation by Targeting NLRP3 Activation at the Trans‐Golgi Network

doi: 10.1002/advs.76587

Figure Lengend Snippet: Pep19‐2.5 reduces pro‐inflammatory IL‐1β response to house dust mite extract in vitro and in vivo. (A–E) Human macrophages from heathy donors were seeded in serum‐free Opti‐MEM medium and stimulated without priming with the indicated doses of house dust mite (HDM) extract for 24 h at 37°C (A) alone, in the presence of (B) the NLRP3 inhibitor MCC950 (10 µM), (C) the TLR4 antagonist eritoran (1 µg/mL), or (D–F) Pep19‐2.5. IL‐1β release was determined from cell‐free supernatants by ELISA. Data shown in (A) mean + SD of duplicate analyses and are representative for n = 7 independent donors. Data in (B–E) were normalized to 100% for HDM in the absence of inhibitors and are shown as mean + SEM of (B, C, E, F): n = 5 and (D): n = 3 independent experiments with cells from different healthy donors. Statistical significance against HDM alone was analyzed by a two‐sided t ‐test. (G) Treatment protocol for HDM‐induced allergic asthma mouse model. (H) Airway resistance in response to methacholine inhalation, (I) inflammatory cell infiltrate volume in lung tissue per epithelial basal membrane (b.m.) area, and (J) numbers of eosinophils on day 14 in bronchoalveolar lavage (BAL) fluid of healthy (PBS), asthmatic (HDM), or Pep19‐2.5‐treated mice (HDM + peptide), n = 10 mice per group. (K–O) Cytokine levels in BAL fluid were assessed by MSD U‐Plex assays. (P) Caspase‐1 mRNA expression in BAL cells and (Q) caspase‐1 protein determined by Western blot. (R) Final body weight on day 14 of the treatment protocol. Results are presented as mean values ± SEM. Statistical significance was assessed using ordinary one‐way ANOVA and Tukey´s multiple comparison post hoc analyses.

Article Snippet: The fluorogenic caspase‐1 substrate Ac‐YVAD‐AMC (HY‐P2717, MedChemExpress, NJ, USA) was added to a final concentration of 10 μM.

Techniques: In Vitro, In Vivo, Enzyme-linked Immunosorbent Assay, Membrane, Expressing, Western Blot, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Epithelial PCSK6 Promotes Proliferation and Decreases Collagen Deposition by Fibroblasts Potentially via MMP Activation

doi: 10.3390/ijms27115104

Figure Lengend Snippet: PCSK6 promotes MMP activation. ( A ) Proteolytic activity of CM from mCherry-control, PCSK6-high and PCSK6-low A549 cells on the fluorogenic MMP substrate peptide {Mca}-Lys-Pro-Leu-Gly-Leu-{Dap(Dnp)}-Ala-Arg-NH2 ( n = 3). ( B ) Proteolytic activity of CM from mCherry-control, PCSK6-high and PCSK6-low A549 cells on the fluorogenic MMP14 substrate peptide MCA-PLA-C(OMeBz)-WAR(Dpa)-NH 2 ( n = 3). ( C ) Proteolytic activity of CM from mCherry-control, PCSK6-high and PCSK6-low A549 cells collected in the presence or absence of 50 µM furin convertase inhibitor (FC inh) on the fluorogenic MMP substrate peptide ( n = 3). ( D ) Proteolytic activity of CM from mCherry-control, PCSK6-high and PCSK6-low A549 cells collected in the presence or absence of 50 µM furin convertase inhibitor (FC inh) on the fluorogenic MMP14 substrate peptide ( n = 3). ( E – G ) Proteolytic activity of CM from mCherry-control, PCSK6-high and PCSK6-low A549 cells in the presence or absence of 500 µM broad-spectrum MMP inhibitor GM6001 ( E ), 50 µM Marimastat ( F ) or the combination of GM6001 and Marimastat ( G ). Fluorescence was measured at excitation 320/20 nm, emission 360/40 nm and normalized to the baseline fluorescence of the substrate peptide in the medium alone. MFI indicates mean channel fluorescence minus background. Data are expressed as mean ± SEM. Statistical significance was tested using mixed-effects analysis with Tukey’s post hoc test for multiple comparisons, and significance is shown relative to the indicated groups. p -values: * <0.05, ** <0.01, *** <0.001 and **** <0.0001.

Article Snippet: MMP activity was assessed using a fluorogenic MMP substrate peptide (CAS 720710-69-0, MCE, Monmouth Junction, NJ, USA), in the presence or absence of the broad-spectrum MMP inhibitor GM6001 (sc-203979, Santa Cruz Biotechnology) or Marimastat (CAS 154039-60-8, MCE).

Techniques: Activation Assay, Activity Assay, Control, Fluorescence

Journal: bioRxiv

Article Title: The microprotein SEP 53BP1 : its bizarre mode of translational expression and intracellular behaviour

doi: 10.64898/2026.05.04.722586

Figure Lengend Snippet: (A). RT-PCR analysis of SEP 53BP1 mRNA expression in the transduced cell lines. The starting material was total cell RNA (quantities are indicated above the panel). (B). A SEP 53BP1 immunoblot prepared from THP-1 transduced cells. A size marker is provided by the SEP 53BP1 synthetic peptide. (C). Immunoblots performed on HeLa, THP1 and macrophages (phorbol 12-myristate 13-acetate (PA) differentiated THP-1 cells) for specific proteasome (β5), immunoproteasome (β5i) and shared markers (20S. 19S. α4). (D). Assays were performed on cell extracts from HeLa and THP-1 cells using the immunoproteasome substrate Ac-Ala-Asn-Trp-AMC (AdipoGen) plus or minus the specific immunoproteasome inhibitor ONX 0914 (AdipoGen). Each time point was monitored in triplicate and plotted graphically as the mean plus the SD. (E). An immunoblot monitoring immunoproteasome induction in HeLa cells lines treated with interferon gamma (IFNγ) or bacterial lipopolysaccharide (LPS) as determined by the expression of β5i (also called LMP7). (F). Immunoproteasome assays performed on the indicated cell lines. (C). This is the same data as presented in panel (B) excluding the THP-1 cell line.

Article Snippet: The immunoproteasome assay was performed in identical buffer conditions but with the fluorogenic peptidyl substrate Ac-Ala-Asn-Trp-AMC (final concentration 10 μM) (AdipoGen) and the specific inhibitor ONX 0914 (10 μM) (AdipoGen) ( , , ).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Marker