EGFR-MET interaction is modulated by EGFR mutations.

<p>(A) Western blot (WB) of total MET levels in H1975 derived cells. Tubulin levels are shown as loading control. Values beneath blots are relative levels of MET compared to the total levels in the H1975^L858R/T790M cell line from 2 independent experiments +/−SD (B) MET (7q31) (red signal) copy number analysis by FISH in the three H1975 cell lines using the Leica Kreatech C-MET (7q31)/SE7 FISH probe (KBI-10719). The green signal indicates the chromosome 7 centromere control probe. Scale bar 10 mm. Average copy number and ratio between MET and chromosome 7 centromere probe are also indicated (n = 30 cells). (C) Immunofluorescence of total EGFR (Alexa546 –red in the image) and MET (Cyanine 5 –green in the image) in H1975 derived cells. Hoescht dye was used to stain the nuclei of the cells. Merge panels are also shown. Bars, 20 μm. (D) Co-immunoprecipitation (IP) of EGFR in H1975 derived cell lines. The EGFR antibody was used to immunoprecipitate. EGFR and MET levels are shown in both bound and input fractions. The gels shown in the figure were run separately for the bound and input fractions, as indicated by the dotted line, under the same experimental conditions. (E) Fluorescence lifetime imaging was performed on cells plated to sub-confluence on cover-slips and time-resolved analysis in Tri2. Quantification of average FRET efficiency (*** p < 0.0005) is shown, as well as representative pseudocolour lifetime images showing FRET efficiency and corresponding grayscale donor (EGFR-Alexa 546) and acceptor (MET-Cyanine 5) intensity images. Scale bar: 50μm.