Chemi reagent mix

Phosphorylation of several kinases and expression levels of MMPs were analyzed using Proteome Profiler Human Phospho-Kinase Array Kit (R&D Systems) and Human MMP Antibody Array Kit (Abcam), respectively, according to the manufacturer's instructions. Briefly, ∼2.0 × 10⁶ cells were grown in complete media for 48 h, lysed, and protein concentration was measured using the BCA protein assay (Pierce). For both arrays, preblocked nitrocellulose membranes were incubated with cellular lysate (corresponding to 500 µg of total protein) o/n at 4 °C and subsequently washed and incubated with biotin-conjugated antibodies and HRP-conjugated streptavidin. Membranes were then incubated with Chemi Reagent Mix and developed in ImageQuant LAS 500 imaging system (GE Healthcare). For protein-level quantification, the integrated optical density (IOD) of each array spot was quantified using ImageJ software (version 1.52i, NIH). IOD values were normalized to those of the positive controls on each membrane and corrected for background signal. Relative expression levels of proteins in two groups of cells were calculated as %. The relative amount of the respective protein in WT was considered as 100%.

Mouse Cytokine Array Panel A kit (CAT# ARY006, R&D Systems, Inc., Minneapolis, MN, USA) was used to detect the relative levels of cytokines and chemokines in mouse plasma, as directed by the manufacturer. Membranes after incubation with the Chemi Reagent Mix (included in the kit) were imaged by using Amersham Imager 600 system (GEHealthcare, Chicago, IL, USA), and a densitometric analysis of the intensities of the cytokine dots was performed by ImageJ software (National Institutes of Health).