Figure 4. Effects of afatinib on protein phosphorylations.

(A-C). Western blot analysis. Cells were pretreated with afatinib for two hours followed by IR. Total cell lysates were collected from PC-9 (A), PC-9-GR (B) and H1975 (C) cells after indicated treatments, and analyzed for phosphorylations of EGFR, AKT and ERK proteins. Anti-GAPDH antibody was included as a loading control. (D). Quantitative analysis for the changes of protein phosphorylations. Densitometry for western blot signal for samples collected two hours post-treatments was conducted, and intensity for the targeted protein/modification was normalized to corresponding GAPDH. Data represent the average results from three independent experiments. (E). RTK array. Cell lysates were collected two hours after IR treatment or IR combined with afatinib, relative phosphorylation level of human receptor tyrosine kinases were determined with Phospho-RTK Array assay as described in Material and Methods.