Quantification of Met Protein Variants

Met, phosphotyrosyl-Met, and soluble Met ectodomain (sMet) proteins were measured in tissue and plasma samples as described previously [22 (link)]. Briefly, streptavidin-coated 96-well plates were blocked, washed with PBS and coated with a biotin-tagged, affinity-purified human Met ectodomain-specific capture antibody (BAF358, R&D Systems). Wells were washed again before adding sample or sMet standard (358-MT, R&D Systems) for 1 h with shaking. After washing with PBS, detection antibody (AF276, R&D Systems) labeled with MSD-Sulfo-Tag (Meso Scale Discovery, Gaithersburg, MD) was added for 1 h with shaking. Wells were washed with PBS before adding read buffer and plates were read in a Meso Scale Discovery SectorImager 2400. Tissue samples were extracted with ice-cold detergent containing buffer with physical disruption with 0.5mm glass beads and shaking in a bead beater (Mini Bead Beater 8, BioSpec Products, Inc., Bartlesville, OK) as described previously [26 (link)]. Assays were performed blinded to study endpoint.