D val leu lys pna 2 hcl

The chromogenic substrate D-Val-Leu-Lys-pNA·2 HCl (Innovative Research) was dissolved in buffer (75 mM Tris HCl, 318 mM NaCl, pH 7.5) (0.25 mM), and treated with human plasminogen in the presence of C. albicans, non-albicans Candida spp. or other recombinant expressed C. albicans cell wall proteins. Urokinase-type plasminogen activator (0.1 g/mL) (Sigma-Aldrich) was used as positive control. The reaction mixture was incubated at 37°C for 24 h and then the absorbance was measured at 405 nm using a multi-mode microplate reader.

IPTG (isopropyl-β-D-thiogalactopyranoside) and DTT were purchased from Sangon Biotech. Ni-nitrilotriacetic acid (Ni-NTA) was purchased from QIAGEN. Human plasminogen, ϵ-aminocaproic acid (ϵ-ACA), Creatinine Assay Kit, glass beads, urokinase-type plasminogen activator, anidulafungin, fluconazole, and DMEM medium were purchased from Sigma-Aldrich. Human Endothelial Serum Free Medium, HuMEC Basal Serum-Free Medium and Blood Urea Nitrogen Detection Kit were purchased from Thermo Fisher Scientific. Mouse nonspecific IgG2a was obtained from Invivogen. Cy3-labelled secondary antibody was purchased from Invitrogen. Chromogenic substrate D-Val-Leu-Lys-pNA·2HCl was purchased from Innovative Research. The LDH Cytotoxicity Assay Kit was obtained from Beyotime. PrimeScript ^TM RT Reagent Kit and the PrimeSTAR® Max DNA Polymerase were obtained from TaKaRa Bio. Rabbit anti-plasminogen antibody was obtained from Acris Antibodies. Anti-actin monoclonal antibody, and horseradish peroxidase (HRP)-conjugated anti-rabbit IgG were obtained from Abcam. HRP-labelled goat anti-mouse antibody was purchased from Dingguochangsheng Biotechnology. pET-21a (+) was purchased from Novagen.