94 Chapter 4 primary) progression, the date of death due to any cause or the cutoff time (set at 60 months). Individuals who were: lost to follow-up or survived beyond the cutoff time, were considered as censored observations. For both survival measures, the cutoff time was set at 60 months. MET protein status and ectodomain shedding disease-free survival as well as overall survival curves were calculated by means of the Kaplan–Meier method and significance of differences in survival times was assessed with the log-rank test. Univariable as well as multivariable Cox proportional hazards regression models were used to evaluate the prognostic value of MET ectodomain shedding, demographical, clinical, and histopathological characteristics. Calculations were performed with SPSS Statistics (version 24; IBM). Unless otherwise mentioned, statistical significance was set at P value < 0.05. Bubble plots used to illustrate the association between N-terminal MET immunoreactivity and survival were generated with Microsoft® Excel (version 2010; Microsoft®; Redmond, WA, USA). Results Reliability and performance of A2H2-3 in relation to D1C2 Under nonreducing conditions, the cell lines HT-29, DU145, PC3, HeLa, and SK-BR-3 showed immunoreactivity with bands migrating as p170 and p145β using this antibody (Fig. 2a). All observed intensities—ranging from weak to strong—were in accordance with measured MET mRNA expression levels. No immunoreactivity was observed for bands migrating otherwise than for MET protein products and NTFs (Supplementary Tables 4, 5). Although A2H2-3 immunoreactivity observed under formalin-fixed paraffin-embedded conditions was heterogeneous, all but one cell line (SK-BR-3) showed scores that are in line with MET mRNA expression levels (Fig. 2b).
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