52 Chapter 3 Before assessing the specificity of the antibodies under reducing conditions, it was assumed that cell lines with low MET mRNA expression levels will show no or weak immunoreactivity with bands migrating as MET protein products and C-terminal fragments (Supplementary table S4). The immunoblots generated with D1C2 and CVD13 (Figure 1B) show band patterns that are specific for MET protein products and C-terminal fragments. Furthermore, the observed intensities are in line with the established MET mRNA expression levels. Moreover, in contrast to its parental cell line (DU145), no immunoreactivity was detected in the MET silenced cell line (DU145#Sh167). When comparing the intensities of the blots generated with D1C2 and CVD13 (Figure 1B), D1C2 shows a stronger immunoreactivity compared to CVD13. This is especially true for the p70MET and p60MET C-terminal fragments observed in HeLa, HT-29 and PC3. In contrast, the immunoblots generated with SP44 and C-12 illustrate that these antibodies are not reliable in detecting of MET protein products and C-terminal fragments (Supplementary Figures S1A & S1B). Although the immunoblot generated with SP44 (Supplementary Figure S1A) only shows immunoreactivity with bands migrating as the expected protein products (Supplementary table S4), the antibody’s performance under reducing conditions was evaluated as nonspecific because of the strong immunoreactivity with a 90 kDa protein product in SK-BR-3 and LNCaP - both cell lines showing low MET mRNA expression levels. C-12’s performance under reducing conditions (Supplementary Figure S1B) was also evaluated as nonspecific, since it shows immunoreactivity with an unexpected 15 kDa protein product in LNCaP, PC3, DU145 and DU145#Sh167. C-12’s nonspecific behavior was further corroborated by moderate immunoreactivity with a 55 kDa protein product in the MET silenced cell line (DU145#Sh167). The immunoblot generated with C-28 was found too poor to evaluate (Supplementary Figure S1C). Taking everything into consideration, it is concluded that only D1C2 and CVD13 specifically detect – at least partly – the expected MET protein products and C-terminal fragments (Supplementary table S4) under reducing conditions. Yet, CVD13 is less sensitive compared to D1C2 (Figure 1B). Under native conditions, immunoreactivities were observed in the nucleus, cytoplasm and at the membrane. Separate scores were given for each cellular compartment. Analogous to the assumptions made under reducing conditions, it was assumed that cell lines with low MET mRNA expression levels will show no or weak immunoreactivity irrespective of the cellular location. This is not the case for C-12 and C-28, which are therefore considered to be nonspecific in the detection of MET (Supplementary Figure S2). However, this is the case for D1C2, CVD13 and SP44 which are therefore considered to be specific in the detection of MET under native
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