rAAV1 and rAAV2 vectors were created by cross-packaging of AAV genomes into AAV1 or AAV2 serotype capsids, respectively . with recombinant adeno-associated disease (rAAV). Methods Cathepsin activity assay Purified pro-catK (human being recombinant), pet cats (human being recombinant) and catB and catH (both from human being liver) (Calbiochem/EMD Millipore, Billerica MA, USA) were used. Pro-catK was triggered at pH 4.0 for 60 min at 25C in NaOAc buffer containing 5 mM DTT and 0.5 mM EDTA. Cathepsins (0.03 nM to 60 nM) were assayed in pH 5.5 MES buffer using a fluorogenic substrate Z-Phe-Arg-amido-4-methylcoumarin (10 to 50 M; Z-Phe-Arg-AMC; Calbiochem) [8,9,23] inside a 96-well plate. The plates were incubated at 25C followed by measurement of fluorescence at Ex lover/Em?=?355/460 nm at various time points. Data were graphed as relative fluorescence devices (RFU) and EC50 ideals were determined by DeltaGraph software (Red Rocks Software, Salt Lake City UT, USA). Inhibition by cysC (human being or mouse; R&D Systems, Minneapolis, MN, USA), trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane) (E-64; Sigma-Aldrich, St Louis, MO, USA) or CA-074 (catB-specific inhibitor; Calbiochem) was YM-264 calculated as percentage of activity compared to no inhibitor present. To quantitate cathepsin activity in rabbit synovium, cells were homogenized using 1mm zirconia beads (Biospec Products Inc, Bartlesville, Okay, USA) in Tper buffer (20 l/mg of cells; Thermo Fisher Scientific, Waltham, MA, USA) and serine YM-264 protease inhibitor cocktail for 30 s followed by chilling in snow for 30 s and repeated five instances. Homogenates were centrifuged and supernatants (30 to 50 ug total protein/sample) were assayed for cathepsin activity using 50 M YM-264 substrate and pH 5.5 as explained above (without low pH activation). Relative activity YM-264 was indicated as RFUs or ideals were normalized by total protein content measured by BCA assay (Thermo Fisher Scientific). rAAV vector production LacZ manifestation cassette contained a human being cytomegalovirus (CMV) promoter, gene and a bovine growth hormone (BGH) polyA. CysC vector contained a cDNA-encoding mouse cysC (GenScript Inc, Piscataway, NJ, USA), a CMV enhancer-chicken -actin promoter and BGH polyA. A negative control, an empty vector (EV), contained CMV promoter and BGH polyA but no transgene. All manifestation cassettes were cloned into a plasmid pAAVSP70 comprising AAV type 2 inverted terminal repeats . rAAV1 and rAAV2 vectors were created by cross-packaging of AAV genomes into AAV1 or AAV2 serotype capsids, respectively . rAAV was generated by triple transfection method and purified using iodixanol columns . Vector titers were quantitated by TaqMan real-time PCR (RT-PCR) using primers and probe specific to the BGH polyA and ideals indicated as DNAse-resistant particles (drp) per ml . cell tradition experiments and protein analyses All enzymes and cells culture reagents were purchased from Invitrogen (Carlsbad, CA, USA). Main chondrocytes and synoviocytes were dissected from rabbit knee bones and digested with ILK (phospho-Ser246) antibody 0.5 mg/ml collagenase at 37C overnight in DMEM followed by culture in DMEM, 10% fetal bovine serum (FBS), 100 IU/ml penicillin and 100 g/ml streptomycin in 5% CO2 incubator at 37C. For catK secretion studies, cells (3 105 cells/well) were plated on 6-well dishes using DMEM press with high glucose, 10% FBS and 50 g/ml gentamicin sulfate. Twelve hours later on, interleukin 1 beta (IL-1) (R&D Systems) was added at 2 ng/ml to some wells. Conditioned press was harvested 48 h later on and concentrated sixfold by centrifugation (Amicon Ultra-5 centrifuge columns (EMD Millipore)) followed by protein analysis. For rAAV illness, synoviocytes and chondrocytes were plated as explained.
rAAV1 and rAAV2 vectors were created by cross-packaging of AAV genomes into AAV1 or AAV2 serotype capsids, respectively