Human protease array kit

After 48 h, supernatants from MRC-5 and hTERT-HGF 2D and 3D cultures were harvested, pooled and filtered through a 100 µm filter (3D condition). Supernatants were centrifuged at 2500 rpm for 5 min to remove insoluble material and stored at −80°C until use. Gels without cells or containing hTERT-HGF were incubated with supernatants from 3D MRC-5 cultures (condition medium, MRC-5 CM) when required. Soluble protease expression profile of MRC-5 and hTERT-HGF 2D and 3D conditions were performed using a Human Protease Array kit according to the manufacturer’s guidelines (R&D Systems, Minneapolis, MI). Images were acquired on a LAS-4000 Fujifilm imager and analyzed after subtraction of background using “Protein Array Analyzer” plugin in (Carpentier and Henault, 2010 ) ImageJ software. The mean pixel density of analyte spots was normalized on reference spots (100%) and negative controls (0%) mean pixel density for each membrane. Results are expressed as Mean Pixel Density (MPD) ± SD. The threshold for significance was inferred from background values and set up at 15%.

In this study, 0.5 mL saliva from each volunteer was tested using a Human Protease Array Kit (R&D Systems, Inc Minneapolis, USA), in accordance with the manufacturer’s instructions. For image analysis, X-ray film (Carestream health Inc. Wuhan, China) was scanned with a CanoScan LiDE 700 F scanner (Canon, Beijing, China) and analyzed by Image J (National Institutes of Health, Bethesda, Maryland, USA). The analysis of each sample was repeated using two array membranes.

The profile of secreted proteases in serum-free S was determined using Human Protease Array Kit (#ARY021, R&D Systems) according to the manufacturer's protocol. Densitometry was performed using ImageJ software (National Institutes of Health) with Protein array analyzer plug-in. The values were normalized to density of 6 reference spots included in each membrane. The list of differentially secreted proteins identified by SILAC LC-MS/MS and antibody arrays was subjected to physical and functional protein associations network search using STRING (http://string-db.org).

Profiling of protein phosphorylation was performed by utilizing a corresponding array (ARY003B; R&D, Minnesota, USA) as per manufacturer’s instruction. Briefly, cell lysates (175 µg total protein) were incubated at 4 °C overnight with the nitrocellulose membranes where capture and control antibodies have been spotted in duplicates. Membranes were washed and incubated with a cocktail of biotinylated detection antibodies for 2 hours at room temperature. Streptavidin-HRP reagent was added to the washed membranes for an additional 30 min interval after which time the membranes were developed with chemiluminescent detection substrate using Image Quant TM LAS 4000 mini machine (GE Healthcare). The signal produced at each capture spot was quantified using ImageJ software and corresponds to the amount of phosphorylated protein bound. The relative protein level of human proteases was assessed using a Human Protease Array Kit (ARY021B; R&D, Minnesota, USA) as per manufacturer’s instruction with the signal captured and quantified essentially as described above.