Journal: The Journal of Biological Chemistry

Article Title: A proteolytic C-terminal fragment of Nogo-A (reticulon-4A) is released in exosomes and potently inhibits axon regeneration

doi: 10.1074/jbc.RA119.009896

Figure Lengend Snippet: The Nogo-66 loop region on the surface of exosomes. A and B, HEK293T cells were transfected with Nogo-A–Myc. 24 h after transfection, media were changed. DMSO, MG101 (20 μm), Z-VAD-fmk (20 μm), E64d (20 μm), and NH4Cl (20 mm) were added and cultured for further 12 h. Then culture supernatants were collected, and the exosome fraction was immunoblotted with anti-Myc and anti-CD9 antibodies (A). The graph shows Myc intensity divided by CD9 intensity compared to DMSO control (B). Mean ± S.E., n = 5–8 independent experiments. *, p < 0.05; ***, p < 0.005; one-way ANOVA followed by Dunnett's test. C and D, HEK293T cells were transfected with Nogo-A–Myc. 24 h after transfection, media were changed. DMSO, the indicated amounts of BACE inhibitors, and NH4Cl (20 mm) were added and cultured for further 12 h. Then culture supernatants were collected, and the exosome fraction was immunoblotted with anti-Myc and anti-CD9 antibodies (C). The graph shows Myc intensity divided by CD9 intensity compared to DMSO control (D). Mean ± S.E., n = 4 independent experiments. **, p < 0.01; ***, p < 0.005; one-way ANOVA followed by Dunnett's test. E, HEK293T cells were transfected with Nogo-A–Myc and siNC, siBACE1 #1, or siBACE1 #2. Exosomes were purified 36 h after transfection and immunoblotted with anti-Myc and anti-CD9 antibodies. F, quantification of Myc intensity divided by CD9 intensity compared to DMSO control. Mean ± S.E., n = 6 independent experiments. **, p < 0.01; ***, p < 0.005; one-way ANOVA followed by Dunnett's test. G, real-time PCR for replicates of siBACE-transfected HEK293T cells. BACE1 mRNA expression was normalized to Gapdh mRNA expression. Mean ± S.E., n = 4 independent experiments. ***, p < 0.005; one-way ANOVA followed by Dunnett's test.

Article Snippet: MG101 (R&D Systems), Z-VAD-FMK (Promega), E64d (Santa Cruz Biotechnology), and β-secretase inhibitor IV (Merck) were used for inhibitor experiments.

Techniques: Transfection, Cell Culture, Control, Purification, Real-time Polymerase Chain Reaction, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Neutrophilic Cathepsin C Is Maturated by a Multistep Proteolytic Process and Secreted by Activated Cells during Inflammatory Lung Diseases *

doi: 10.1074/jbc.M115.707109

Figure Lengend Snippet: Processing of proCatC in PLB-985 cells cultured in the presence or absence of synthetic inhibitors. A, cell surface CD11b expression was analyzed by flow cytometry. Undifferentiated PLB-985 were cultured for 1 week in the presence of ICatS (10 μm), ICatC (2 μm), or after adding the DMSO/DMF containing buffer alone. Cell lysates were analyzed by Western blotting using anti-CatC antibody (Ab1). The percentage of CatC, PR3, and CatG activity was determined using the respective selective substrates for each protease and is given in the box. B, PLB-985 cells were differentiated into neutrophil-like in the presence of DMF and with or without ICatS (10 μm) or ICatC (2 μm) or DMSO/DMF. Cell lysates were analyzed by Western blotting using anti-CatC antibody (Ab1). The % of CatC, PR3, and CatG activity measured using their respective selective substrates were shown in the box. Cell surface CD11b expression was analyzed by flow cytometry as in A. C, undifferentiated PLB-985 cells cultured in the presence of E64c (100 μm), E64d (100 μm), or DMSO alone for 48 h were lysed. Cell lysates and supernatants of PLB-985 cells were analyzed by Western blotting using anti-CatC antibody Ab1 (left and right panels, respectively). Similar results were observed in five independent experiments.

Article Snippet: E64c ((2 S ,3 S )- trans- epoxysuccinyl- l -leucylamido-3-methylbutane) and E64d (2 S ,3 S-trans -(ethoxycarbonyloxirane-2-carbonyl)- l -leucine-(3-methylbutyl) amide) were from Santa Cruz Biotechnology.

Techniques: Cell Culture, Expressing, Flow Cytometry, DMSO/DMF Sterilization, Western Blot, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: Neutrophilic Cathepsin C Is Maturated by a Multistep Proteolytic Process and Secreted by Activated Cells during Inflammatory Lung Diseases *

doi: 10.1074/jbc.M115.707109

Figure Lengend Snippet: Processing of proCatC in HL-60 cells cultured with or without synthetic inhibitors. A, HL-60 cells were cultured for 1 week in medium containing ICatS (10 μm), ICatC (2 μm), ICatS/ICatC (10 μm/2 μm), DMF, or DMSO/DMF, and then total cell lysates were analyzed by Western blotting using anti-CatC antibody (Ab1). The percentages of residual CatC, PR3, and CatG activity toward their respective selective substrates were given in the box. B, HL-60 cells were cultured for 1 week in medium containing ICatS (10 μm), ICatC (2 μm), and ICatS/ICatC inhibitors (10 μm/2 μm), and their lysates were analyzed by Western blotting using anti-CatC antibody (Ab2). C, HL-60 cells were cultured for 48 h in medium containing ICatS (10 μm), E64c (100 μm), or E64d (100 μm), and then total cell extracts were analyzed by Western blotting using anti-CatC antibody (Ab1). Similar results were observed in five independent experiments. D, diagram summary showing the processing of proCatC in HL-60 cells cultured in the presence of ICatS or E64d. Arrows indicate the position of proteolytic cleavages in proCatC. CP, cysteine protease. E, partial sequence of the CatC propeptide (residues 1–65) showing the N-terminal proteolysis-sensitive extension (residues 1–24) and the structurally conserved sequence found in CatL-like cysteine proteases (residues 24–65). Model structure was obtained using cysteine peptidase C (PDB code 4HWY; Ref. 37) as a template.

Article Snippet: E64c ((2 S ,3 S )- trans- epoxysuccinyl- l -leucylamido-3-methylbutane) and E64d (2 S ,3 S-trans -(ethoxycarbonyloxirane-2-carbonyl)- l -leucine-(3-methylbutyl) amide) were from Santa Cruz Biotechnology.

Techniques: Cell Culture, DMSO/DMF Sterilization, Western Blot, Activity Assay, Sequencing

Journal: Journal of Medical Virology

Article Title: Native and activated antithrombin inhibits TMPRSS2 activity and SARS‐CoV‐2 infection

doi: 10.1002/jmv.28124

Figure Lengend Snippet: Antithrombin inhibits activity of cathepsin L, while moderately affecting cathepsin B. Recombinant cathepsin L (A) or isolated cathepsin B (B) were incubated with AT (Anbinex), small molecule TMPRSS2 inhibitor CM or small molecule cathepsin inhibitor E64‐d, 1 h before the addition of fluorogenic substrate Z‐L‐R‐AMC (for cathepsin L) or Z‐R‐R‐AMC (for cathepsin B). Data are shown as means ± SEM derived from n = 3 experiments performed in triplicates. AT, antithrombin; CM, camostat mesylate; SEM, standard error of the mean.

Article Snippet: For analysis of purified TMPRSS2, serially diluted AT, activated AT, CM, or E64‐d were mixed with 25 µl of 2 µg/ml recombinant TMPRSS2 in assay buffer (50 mM Tris‐HCl, 0.154 mM NaCl pH 8.0) for 10 min at 37°C, followed by addition of 50 µl of 20 µM BOC‐QAR‐AMC protease substrate.

Techniques: Activity Assay, Recombinant, Isolation, Incubation, Derivative Assay