Figure 6

Knockdown by RNAi qualifies EGFR involvement in ME180 AP-1 CellSensor pathway. AP-1-bla ME180 cells transfected with RNAi targeting EGFR were analyzed by RT-qPCR (Panel A). EGFR mRNA expression is knocked down ~80% as compared to a non-specific control (Med GC). Results are represented as a ratio using cyclophilin as a control. Cells RNAi treated with oligos specific for EGFR show a reduced level of EGFR protein on the cell surface including a loss of autophosphorylation (Panels B and C). RFU represent measurement of the Alexa fluor 488 and 594 labelled secondary antibodies used for detection of the phospho and pan EGFR antibodies and represent the mean of (± SEM) eight independent data points (n = 8). RNAi treated AP-1-bla ME180 cells also exhibit a knockdown of AP-1 activation when EGF stimulated and analyzed at different timepoints (Panel D). Normalized response represents the amount of AP-1 gene activation present when compared with a non-specific control (Med GC), and represent the mean of (± SEM) eight independent data points (n = 8).