Figure 2.

Influence of NES mutation on EGFR kinase activity and nuclear translocalization. (A) Transfection of EGFR WT and mutants was performed and the transfected cells were serum-starved for 20 h and stimulated with 50 ng/ml EGF for 30 min. The precipitates with anti-myc antibodies were immunoblotted with anti-phospho-tyrosine antibodies (4G10) and anti-EGFR. (B) HEK293T cells were transfected with EGFR^WT and EGFR^L747A mutant, starved for 20 h, and then treated with or without EGF for 30 min. A Western blot analysis was performed with specific antibodies against myc (EGFR), phospho-STAT3 (p-STAT3), and STAT3. (C) HEK293T cells were transfected with the indicated plasmids. The transfected cells were starved for 20 h and pretreated with the EGFR inhibitor erlotinib at 10 µM for 30 min, followed by EGF stimulation for 30 min. The cell fractions were separated and immunoblotted with the indicated antibodies. (D) HEK293T cells were transiently transfected with the indicated plasmids. EGF treatment and analysis were performed as described in (C). (E) H1299 cells with endogenous knockdown EGFR were reconstituted with EGFR WT, L747, and L858R mutants. The stable cell lines were starved for 16 h and stimulated with 100 ng/ml EGF, with or without pretreatment with erlotinib (10 µM) for 45 min. Images were obtained using confocal microscopy. Bar, 10 µm. (F) Semi-quantitative analysis of confocal images. Fractions of EGFR co-localized with DAPI (inside the nucleus) pixels vs. total EGFR pixels at the confocal plane were calculated using LSM710 software. Data were compiled from three independent experiments. Nuclear EGFR is represented as the ratio of nuclear vs. total EGFR intensities. ns, not significant, *P < 0.05.