fluorogenic substrate 4 methylumbelliferyl 2 acetamido 2 deoxy α d glucopyranoside  (Biosynth Carbosynth)

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

Journal: bioRxiv

Article Title: Discovery, characterisation and optimisation of bicyclic peptide inhibitors that disarm Staphylococcus aureus α-hemolysin

doi: 10.64898/2026.03.09.710508

Figure Lengend Snippet: a) Alexa Fluor 647-labeled AhlyH35A (7.5nM) was incubated with A549 cells in the presence of increasing concentrations of Peptide 88 or a control bicyclic peptide. Cell-associated fluorescence was quantified by flow cytometry and shown as histogram overlays. Negative control (cells only) shown in black; positive control (AhlyH35A without peptide) shown in red. b) Quantification of median fluorescence intensity plotted against peptide concentration. Data are normalized to the negative and positive controls. c) ADAM10 protease activation by Ahly (6µM) was measured using a whole-cell FRET peptide cleavage assay in the presence of Peptide 88 or a control bicyclic peptide (900µM). Mean of two biological replicates; error bars indicate standard deviation. Data were analysed using one-way ANOVA with Dunnett’s test: ns = not significant; ** = P < 0.01.

Article Snippet: Following incubation, cells were washed once with 25mM Tris buffer, pH 8.0 and a fluorogenic ADAM10 substrate peptide (Mca-PLAQAV-Dpa-RSSSR-NH 2 ; R&D Systems) was added at a final concentration of 10μM.

Techniques: Labeling, Incubation, Control, Fluorescence, Flow Cytometry, Negative Control, Positive Control, Concentration Assay, Activation Assay, Cleavage Assay, Standard Deviation