selective cathepsin b inhibitor  (MedChemExpress)

Journal: Frontiers in Plant Science

Article Title: Anionic Phospholipids Induce Conformational Changes in Phosphoenolpyruvate Carboxylase to Increase Sensitivity to Cathepsin Proteases

doi: 10.3389/fpls.2019.00582

Figure Lengend Snippet: (A) sp-PEPC exposed to PA in the presence of protease inhibitors: sp-PEPC control (lane 1), no protease inhibitors (lane 2), 1 mM Bestatin (lane 3), 0.2 mM Leupeptin (lane 4), 0.065 mM Chymostatin (lane 5), 1 mM PMSF (lane 6), Sigma protease inhibitor cocktail containing AEBSF, 1,10-Phenanthroline, Pepstatin, Leupeptin, Bestatin and E-64 (lane 7) or 0.1 mM Aprotinin (lane 8). Following 20 h of incubation at 30°C, samples were subjected to SDS–PAGE and stained with Commassie Blue. (B) Cistatin and E-64 inhibit the protease activity present in sp-PEPC. Aliquots of sp-PEPC (25 μl) were incubated with 1 μM of recombinant purified cystatin (CIP) or E-64 in a 0.1 M citrate buffer (pH 6), 0.15 M NaCl and 5 mM MgCl 2 for 10 min at 30°C. Protease activity was assayed by addition of 2 mM of the cathepsin L-specific substrate, Z-FR-AMC. The reaction was developed 5 h at 30°C. Protease activity is expressed as % with respect to the control in absence of inhibitor (sp-PEPC). Date are the mean ± SE of triplicate measurement and statistically significant difference with respect to the control are indicated by ( ∗ ) at P < 0.05, ( ∗∗ ) at P < 0.01, using the Dunnett test.

Article Snippet: Protease inhibitors used were: Chymostatin (cysteine protease, chymotrypsin and elastase inhibitor); phenylmethane sulfonyl fluoride (PMSF; serine protease inhibitor); Aprotinin (serine protease inhibitor); Bestatin (aminopeptidase inhibitor); Leupeptin (serine and cysteine protease inhibitor); E-64 (selective cysteine protease inhibitor, such as cathepsin B and L); and protease inhibitor cocktail (AEBSF; serine proteases inhibitor; Phenanthroline, metalloproteases inhibitor; Pepstatin A, acid proteases inhibitor, Leupeptine, Bestatin and E-64) from Sigma Aldrich code P9599 (St Louis, MO, United States).

Techniques: Protease Inhibitor, Incubation, SDS Page, Staining, Activity Assay, Recombinant, Purification

Journal: PLoS ONE

Article Title: Cobalt Alloy Implant Debris Induces Inflammation and Bone Loss Primarily through Danger Signaling, Not TLR4 Activation: Implications for DAMP-ening Implant Related Inflammation

doi: 10.1371/journal.pone.0160141

Figure Lengend Snippet: IL-1β and TNF-α was assessed after (A-B) THP-1 differentiated macrophages and (C-D) THP-1 monocytes were challenged with Cobalt-alloy particles (particles:cell = 5:1), Cobalt-alloy/LPS+, and LPS in the presence of either PAb isotype matched control antibody (4μg/mL), PAb-hTLR4 (4μg/mL), or Cathepsin-B inhibitor (10uM) for 20 hours. Cathepsin B danger signal (inflammasome) blocking for Cobalt alloy significantly decreased IL-1β responses to all challenges, more than TLR blocking in both macrophages (A-B) and monocytes (C-D). Note: */** p<0.05 to each treatment groups respective control values

Article Snippet: LPS (TLR4 positive control), PAb Control (control isotype) and PAb-hTLR4 (polyclonal antibody to human TLR4), Z-VAD-FMK (inhibitor of caspase-1 activation in NLRP3 induced cells) (InvivoGen), and selective lysosomal Cathepsin-B inhibitor (CA-074-Me) (Sigma-Aldrich) were used in challenged monocytes/macrophages.

Techniques: Blocking Assay