Journal: Molecular cell

Article Title: Methyl-Metabolite Depletion Elicits Adaptive Responses to Support Heterochromatin Stability and Epigenetic Persistence

doi: 10.1016/j.molcel.2020.03.004

Figure Lengend Snippet: Data and Software Availability

Article Snippet: Leupeptin Hemisulfate , Thermo Fisher , Cat#AAJ61188MC.

Techniques: Software, Recombinant, Sequencing, Modification, Extraction, Reverse Transcription, Multiplex sample analysis, Plasmid Preparation, SYBR Green Assay, Staining, cDNA Synthesis, DNA Purification, Purification, Control, Targeted Proteomics

Journal: Molecular cell

Article Title: Methyl-Metabolite Depletion Elicits Adaptive Responses to Support Heterochromatin Stability and Epigenetic Persistence

doi: 10.1016/j.molcel.2020.03.004

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Leupeptin Hemisulfate , Thermo Fisher , Cat#AAJ61188MC.

Techniques: Recombinant, Sequencing, Modification, Extraction, Reverse Transcription, Multiplex sample analysis, Plasmid Preparation, SYBR Green Assay, Staining, cDNA Synthesis, DNA Purification, Purification, Control, Software, Targeted Proteomics

Journal: The Journal of biological chemistry

Article Title: NPC1 plays a role in the trafficking of specific cargo to melanosomes.

doi: 10.1016/j.jbc.2023.105024

Figure Lengend Snippet: Figure 5. NPC1 impairs TYR processing and degradation. A, cells were untreated or treated with bafilomycin A (100 nM, 5 h) and analyzed by Western blotting for TYR N-glycan processing after Endo H and PNGase F glycosidase treatment of cell lysates. Relative expression levels of PNGase F-sensitive TYR and the ratio of Endo H-resistant to Endo H-sensitive TYR were plotted. Quantitative representation as mean ± SD of relative protein expressions and the ratio of Endo H resistant to Endo H sensitive bands (one-way ANOVA analysis; *p < 0.05, **p < 0.01, n = 3 biological replicates). B, visual comparison of cell pellets of MNT-WT and NPC1-KO cells untreated or treated with bafilomycin A1 (100 nM, 5 h). C, immunoblot analysis for TYR, LAMP-2, and CNX in cell lysates and in the enrichment of secreted extracellular vesicles from culture supernatant. D, effect of MG132 (50 μM, 5 h) or inhibitor mix (INH = 14.5 uM leupeptin and 106 uM pepstatin, 5 h) cell treatment on TYR expression, analyzed by Western blotting and densitometry, represented as fold change relative to cells treated with DMSO. Quantitative representation as mean ± SD of relative protein expression, n = 3 biological replicates (one-way ANOVA analysis; *p < 0.05). E, CHX treated MNT-WT and NPC1-KO cells analyzed by immunoblotting for TYR normalized to tubulin and graphic representation of TYR degradation rate as a percentage of control (CHX, 0 h) (n = 2 biological replicates). CHX, cycloheximide; CNX, calnexin; DMSO, dimethyl sulfoxide; Endo H, endoglycosidase H; LAMP, lysosome-associated membrane protein; NPC1, Niemann–Pick type C1; PNGase F, peptide-N glycosidase F; TYR, tyrosinase.

Article Snippet: MG132 (sc-201260), leupeptin hemisulfate (sc295358) and pepstatin A (sc-45036) were from Santa Cruz Biotechnology.

Techniques: Western Blot, Glycoproteomics, Expressing, Comparison, Control, Membrane

Journal: mBio

Article Title: Mechanism of Microbial Metabolite Leupeptin in the Treatment of COVID-19 by Traditional Chinese Medicine Herbs

doi: 10.1128/mBio.02220-21

Figure Lengend Snippet: Leupeptin can inhibit the catalytic activity of SARS-CoV-2 M pro in vitro . (A) Chemical structure of leupeptin hemisulfate. (B) Inhibitory curve of leupeptin hemisulfate against SARS-CoV-2 M pro . (C) Overall structure of SARS-CoV-2 M pro dimer in complex with leupeptin. (D) Detailed interactions between leupeptin (yellow) and SARS-CoV-2 M pro . (E) Binding pocket of leupeptin bound to Carica papaya papain protease (PDB accession number 1POP ). (F) Binding pocket of leupeptin bound to human calpain-3 protease (PDB accession number 6BKJ ).

Article Snippet: A total of 20 mM leupeptin hemisulfate (catalog number T6564; TargetMol) in deionized water was diluted to 2 mM to 31.25 μM with 25 mM Tris buffer (pH 8.0).

Techniques: Activity Assay, In Vitro, Binding Assay

Journal: mBio

Article Title: Mechanism of Microbial Metabolite Leupeptin in the Treatment of COVID-19 by Traditional Chinese Medicine Herbs

doi: 10.1128/mBio.02220-21

Figure Lengend Snippet: Inhibition of SARS-CoV-2 replication in Vero cells by leupeptin. (A) Inhibitory curve of leupeptin against SARS-CoV-2 replication. (B) Relative concentration of vRNA present in the supernatant at 72 h postinfection determined by reverse transcription-quantitative PCR (qRT-PCR) analysis after leupeptin treatment. Continued t test P values indicate statistical significance (**, P < 0.01; ***, P < 0.001).

Article Snippet: A total of 20 mM leupeptin hemisulfate (catalog number T6564; TargetMol) in deionized water was diluted to 2 mM to 31.25 μM with 25 mM Tris buffer (pH 8.0).

Techniques: Inhibition, Concentration Assay, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

Journal: mBio

Article Title: Mechanism of Microbial Metabolite Leupeptin in the Treatment of COVID-19 by Traditional Chinese Medicine Herbs

doi: 10.1128/mBio.02220-21

Figure Lengend Snippet: HPLC and HRMS analyses of leupeptin and the QFPD decoction. (A) HPLC chromatogram of the leupeptin reference standard. (B) HPLC chromatogram of the QFPD decoction. The chromatographic peaks in boxes with dotted lines indicate the probable leupeptin substance in the QFPD decoction. (C) MS/MS spectrum of the precursor ion ( m/z 427.2969) in the leupeptin reference standard. (D) MS/MS spectrum of the precursor ion ( m/z 427.2968) in the QFPD decoction. (E) Proposed fragmentation pathway of leupeptin. The numbers 1 to 11 represent the different fragments of leupeptin.

Article Snippet: A total of 20 mM leupeptin hemisulfate (catalog number T6564; TargetMol) in deionized water was diluted to 2 mM to 31.25 μM with 25 mM Tris buffer (pH 8.0).

Techniques: Tandem Mass Spectroscopy

Journal: mBio

Article Title: Mechanism of Microbial Metabolite Leupeptin in the Treatment of COVID-19 by Traditional Chinese Medicine Herbs

doi: 10.1128/mBio.02220-21

Figure Lengend Snippet: Plant-symbiotic actinomyces and antiviral activity ecology of microbial bioactive compounds, such as leupeptin, contribute to the antiviral activity of the QFPD decoction against SARS-CoV-2 M pro .

Article Snippet: A total of 20 mM leupeptin hemisulfate (catalog number T6564; TargetMol) in deionized water was diluted to 2 mM to 31.25 μM with 25 mM Tris buffer (pH 8.0).

Techniques: Activity Assay