Figure 6. Phosphorylation of EGFR at Y1101 is involved in the nuclear localization of EGFR.

(A) Immunoprecipitation of total EGFR followed by immunoblotting with anti-pEGFR Y845, anti-pEGFR Y1101 or pan-EGFR antibodies. MCF7 Cells overexpressing EGFR-WT, EGFR-Y845F or EGFR-Y1101F were harvested after stimulation with 100 ng/mL of EGF for 45 min. A total of 500 ug of cell lysate was immunoprecipitated with pan-EGFR antibody. The immunoprecipitates were fractionated on SDS-PAGE followed by immunoblotting for the indicated proteins. (B) Mutation of Y1101, but not Y845, reduces the nuclear localization of EGFR in MCF-7 breast cancer cells. Cells were transiently transfected with plasmids encoding the EGFR wild-type (WT), EGFR-Y845F, EGFR-Y1101F or vector only. 48hr post-transfection the cells were incubated with EGF (100 ng/ml) for 45min, harvested for whole cell lysate, non-nuclear, and nuclear protein, and fractionated on SDS-PAGE followed by immunoblotting for indicated proteins. Histone H3 and α-tubulin were used as loading and purity controls for the nuclear and non-nuclear fractions, respectively. (C) B-Myb and iNOS mRNA levels were down-regulated in HC4 cells transfected with EGFR-Y1101F mutant compared to EGFR-WT transfected cells by qPCR. Cells were transiently transfected with plasmids encoding the EGFR wild-type (WT) or EGFR-Y1101F. 24hr post-transfection the cells were treated with EGF (100 ng/ml) for 45min, and harvested for RNA. The mRNA expression of B-Myb and iNOS was determined by qPCR.