Magic red cathepsin l assay kit

Enzyme activity assays were performed in J774A.1 cells. For Cathepsin C enzyme activity; we used Gly-Phe β-naphthylamide as a substrate. Lysosomes of J774A.1 cells were pre-labeled with TMR-dextran (0.5 mg/mL; G) for 1 hr and chased in complete medium for 16 hr at 37°C. The cells were then labeled with 50 nM LysoTracker in complete medium for 30 mins at 37°C. 50 μM NPPB or 200 μM GPN were then added to the cells and incubated for 30 mins at 37°C. The cells then washed and imaged in HBSS buffer containing either NPPB or GPN. The whole cell intensity ratio (G/R) was plotted to quantify the level of LysoTracker labelling of the endosomes. For Cathepsin L and Aryl Sulfatase enzyme activity Magic Red Cathepsin L assay kit (Immunochemistry Technologies) and Lysosomal sulfatase assay kit (Marker Gene) were used. The experiment was performed using the manufacture’s protocol. Briefly, cells were incubated with 1X Magic Red Cathepsin L assay probe or 200 μM Lysosomal sulfatase assay probe for 4 hr in complete medium. The cells were then labelled with 10 μM Hoechst stain for 10 mins at 37°C after which the cells were washed and imaged. For low chloride containing dishes; cells were preincubated with 50 μM NPPB before the addition of the enzyme probes. The ratio of enzyme substrate whole cell intensity to that of DAPI was used to quantify enzyme activity.

A rabbit anti-PEDV-N protein polyclonal antibody was generated in the laboratory [24 (link)]. A beta-actin rabbit polyclonal antibody was purchased from Proteintech Group, Inc. (Rosemont, IL, USA). Alexa 488-conjugated goat anti-rabbit IgG antibody and 4’,6-diamidino-2-phenylindole (DAPI) were obtained from Thermo Fisher Scientific, Inc. (San Hose, CA, USA). The Cell Counting Kit-8 (CCK-8) was purchased from Apex Bio Technology, Inc. (Houston, TX, USA). A soluble TMB Substrate Solution (TMB) was obtained from Tiangen (Beijing, China). HiScript II Reverse Transcriptase and 2×Taq Master Mix were purchased from Vazyme Biotech Co., Ltd. (Nanjing, China). An RNA extraction kit was obtained from PuDi Biotech Co., Ltd. (Shanghai, China). LysoSensor™ Yellow/Blue DND-160 was purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Magic Red^® Cathepsin L Assay Kit and Magic Red^® Cathepsin B Assay Kit were obtained from Immunochemistry Technologies, LLC. (Davis, CA, USA). BBM, FAN, and +FAN were purchased from Selleck Chemicals (Houston, TX, USA). NH4Cl was obtained from Sigma-Aldrich (St. Louis, MO, USA).