Specific cleavage at the FKFL linker by cathepsin B was determined by adapting a method from Dubowchik et al.44 and Chu et al.47 Human liver cathepsin B (Enzo Life Sciences) was activated for 15 min in a solution of 0.158 mg/mL cathepsin B, 20 mM DTT, and 10 mM EDTA at 37°C. Peptide or polymer was then solubilized in reaction buffer (10 mM phosphate, 1 mM EDTA, pH 6.6, 37°C) and added to the enzyme solution for a final concentration of 1.28 μg/mL cathepsin B and 65 μM peptide/polymer. At various time points, reaction aliquots were removed, enzymatic activity was halted by addition of a thioprotease inhibitor (E-64 (Thermo Scientific), 26 μg/mL), and RP-HPLC and mass spectrometry were used to quantify cathepsin B cleavage of the FKFL linker. Cleavage of the polymers was also visualized by SDS–PAGE on 8–16% Tris-glycine gels (Bio-Rad). For protein gel analyses, reactions were conducted at 3× concentration and 160 μg of polymer was loaded per well.
Cathepsin b
Manufactured by Thermo Fisher Scientific
Cathepsin B is a lysosomal cysteine protease that is involved in intracellular protein degradation and turnover. It plays a role in various physiological and pathological processes.
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Lab products found in correlation
2 protocols using cathepsin b
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Cathepsin B-Mediated Cleavage Assay
2
Western Blot Analysis of Alzheimer's Markers
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CHO-7WD10 and SH-SY-5Y cells were seeded at 1 × 106 cells/well in a six-well plates and stimulated with various concentrations of PGDHC and Pg-LPS as listed for the cytotoxicity assay. After 48 h of stimulation, cells were lysed in the lysis buffer (Thermofisher) and protein concentration was measured using the BCA kit (Pierce). Next, proteins were separated using SDS/PAGE (Bolt 12% gel) electrophoresis, transferred onto a nitrocellulose (NC) membrane and blocked using iBlot2 (Thermofisher). The anti-mouse CT15 polyclonal antibody (1:500) (Calbiochem) was used for detection of full-length APP in CHO-7WD10.
To detect phosphorylated-Tau (p-Tau), sirtuin-1, and cathepsin B in SH-SY5Y cells, rabbit anti-p-Tau (Ser396), and -mouse AT1000 (Thr212/Ser214), - Sirt-1 and -cathepsin B polyclonal antibodies (1:1,000; Thermofisher) were used, respectively. The anti-human β-actin antibody (cat # 1:2,000; CST) was used to detect the levels of β-actin as a loading control. Finally, the membranes were washed with tris‐buffered saline (TBS) containing 0.05% Tween 20 and then processed using horseradish peroxidase (HRP)‐conjugated anti‐rabbit or anti‐mouse secondary antibodies (Amersham Pharmacia Biotech) followed by enhanced chemiluminescence detection (ThermoFisher). The signal intensity of Western blots was quantified using Image J.
To detect phosphorylated-Tau (p-Tau), sirtuin-1, and cathepsin B in SH-SY5Y cells, rabbit anti-p-Tau (Ser396), and -mouse AT1000 (Thr212/Ser214), - Sirt-1 and -cathepsin B polyclonal antibodies (1:1,000; Thermofisher) were used, respectively. The anti-human β-actin antibody (cat # 1:2,000; CST) was used to detect the levels of β-actin as a loading control. Finally, the membranes were washed with tris‐buffered saline (TBS) containing 0.05% Tween 20 and then processed using horseradish peroxidase (HRP)‐conjugated anti‐rabbit or anti‐mouse secondary antibodies (Amersham Pharmacia Biotech) followed by enhanced chemiluminescence detection (ThermoFisher). The signal intensity of Western blots was quantified using Image J.
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