53 MET immunoreactivity and poor prognosis conditions. The specific behavior of these three antibodies is further supported by the absence of immunoreactivity in the MET silenced cell line (DU145#Sh167). In contrast to D1C2 and CVD13, SP44 shows weak immunoreactivity with SK-BR-3 and LNCAP making it the most sensitive antibody under native conditions (Figure 1C, Supplementary Figure S2). Also under FFPE conditions, immunoreactivities were observed in the nucleus, cytoplasm and at the membrane. Again, separate scores were given for each cellular compartment and – similar to the assumptions made under native conditions – it was assumed that cell lines with low MET mRNA expression levels will show no or weak MET immunoreactivity irrespective of the cellular location. This is not the case for SP44, C-12 and C-28, which are therefore considered to be nonspecific in the detection of MET under FFPE conditions (Supplementary Figure S3). However, this is the case for D1C2 and CVD13, which are therefore considered to be specific in the detection of MET in FFPE cells. This is further supported by the weak immunoreactivity observed in the MET silenced cell line (DU145#Sh167). Comparing the staining intensities obtained with D1C2 and CVD13 per subcellular localization, reveals that D1C2 has a higher sensitivity for membranous MET and that CVD13 has a higher sensitivity for cytoplasmic MET in FFPE cells (Figure 1D). To verify whether the latter observation holds true in a diagnostic setting, four routinely processed FFPE oral SCC were stained with D1C2 and CVD13. Again, D1C2 shows a higher sensitivity for membranous MET and CVD13 shows a higher sensitivity for cytoplasmic MET. This is especially true for immunoreactivities observed with salivary gland ducts and cancer cells (Supplementary Figures S4A & S4C). The specificity of D1C2 was corroborated for all examined conditions by performing western blot analysis, immunocytochemistry and immunohistochemistry on all examined cell lines and cancers after pre-incubating the antibody with the peptide that was used for its generation (Figure 2). 3
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