88 Chapter 4 were HeLa, PC3, and HT-29. LNCaP served as negative control. MET (Hs01565584_ m1; Life TechnologiesTM/Applied Biosystems®, Grand Island, NY, USA) and HPRT1 (Hs02800695_m1; Life TechnologiesTM) mRNA expression levels were determined at a threshold of 0.2. Next, the expression levels of MET was standardized using the expression levels of HPRT1 according to relative standard curve method (4371095 Rev A; Life TechnologiesTM). Average raw Ct values for MET and HPRT1 can be found in Supplementary Table 3. MET antibodies Western blotting, immunohistochemistry and ELISA were performed using antibodies directed against the C- and/or N-terminus of MET. More specifically, D1C2 (Cell Signaling Technology®; Danvers, MA, USA) and A2H2-3 (Eli Lilly and Company; Indianapolis, USA). Further details concerning the antibodies are described in (18) for D1C2 and (28) for A2H2-3. Protein isolation and western blot analysis using D1C2 Culturing of the cell lines, protein isolation, and western blot analysis using D1C2 under reducing conditions were performed as previously described (18). Intensities of the relevant bands were evaluated as follows: absent, weak, moderate, and strong. Representative images can be found in the main text. Assessment of MET ectodomain shedding and regulated intramembrane proteolysis using D1C2 under reducing conditions For protein isolation, representative HE sections of the included fresh frozen tissues were used to verify if the tumor/stroma ratio of the analyzed samples was above 50%. Protein isolation and western blot analysis were performed using the same protocol as used for the cell lines. Together, HeLa, PC3, and HT-29 served as a possible control for full length MET, MET C, and N-terminal fragments resulting after ectodomain shedding and regulated intramembrane proteolysis (Supplementary Tables 4, 5). Since all C- and N-terminal fragments are derived for the mature MET protein product, it is assumed that the intensities of C-terminal fragments under reducing conditions should be similar or weaker to the intensity observed for p145β within samples. Protein isolation and western blot analyses using A2H2-3 Cells were cultured until 75–90% confluence, scraped in HBSS (Life TechnologiesTM), and frozen at −80 °C. Tumor cells were then lysed and immunoblotted with
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