Fig. 2

NEDD4 is associated with activated EGFR. a, Co-immunoprecipitation of NEDD4 with activated EGFR in lung cancer cells. Lung cancer A549 or H358 cells were serum-starved for 12 h followed by stimulation with EGF (50 ng/ml) for indicated times. EGFR was immunoprecipitated with anti-EGFR (Mab528) and detected by immunoblotting with anti-EGFR (1005) (top panels). Co-immunoprecipitated NEDD4 was detected by immunoblotting with an anti-NEDD4 (second top panels). The level of EGFR and NEDD4 in the cell lysates was also detected by immunoblotting (middle and second bottom panels). Notice that EGFR in A549 and H358 cells has an EGF-induced degradation. b, Internalized EGFR is co-localized with NEDD4. A549 cells were serum-starved for 12 h followed by stimulation with EGF (50 ng/ml) for 0 or 60 min. The cells were immuno-stained with anti-EGFR (1005) (red) and anti-NEDD4 (green). Bar, 20 μM. c, Co-expression of NEDD4 with EGFR in lung adenocarcinoma tissue. The tissue microarray containing 63 lung adenocarcinoma section samples was immunohistochemically stained with anti-EGFR or anti-NEDD4