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: The FEBS journal

Article Title: Verapamil treatment induces cytoprotective autophagy by modulating cellular metabolism.

doi: 10.1111/febs.14064

Figure Lengend Snippet: Fig. 7: Verapamil induces autophagic flux Western blots for LC3 protein levels after treatment with increasing concentrations of Verapamil (Ver; 50, 100, 200 µM; 6 h) alone or in combination with lysosomal protease inhibitors – E64d/Pepstatin A (E/PepA; 10 µM; 6 h) (A-E). Actin or ERK2 were used as a loading control. Representative images of the Western blots are shown (upper panels) together with the quantification of LC3-II fold increase in comparison to control, untreated cells (lower panels). Each performed experiment is presented as an individual data point set with mean (±SEM), n= 3 to 6.

Article Snippet: Verapamil chloride (Sigma-Aldrich, V4629), E64d (LKT Laboratories, E0003), Pepstatin A (Calbiochem, 516481), Chloroquine (Sigma-Aldrich, C6620), Sodium oxamate (Sigma-Aldrich, O2751).

Techniques: Western Blot, Control, Comparison