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. 2012 Oct 1;5(5):379–392. doi: 10.1593/tlo.12235

Figure 1.

Figure 1

EGFR knockdown by specific shRNA reduces c-Met expression. (A) Down-regulation of EGFR by an EGFR-specific shRNA results in loss of c-Met phosphorylation in both U251 and 5310 cells when transfected for 72 hours. Corresponding whole-cell lysates were immunoblotted with anti-Met, anti-Stat3, and β-catenin antibodies. GAPDH served as a loading control (SV, scrambled vector). (B) Quantitation of A. (C) U251 and 5310 cells were serum starved for 3 hours and then treated with 10 ng/ml rEGF for 1 hour. Total and phosphorylated EGFR, c-Met, Stat3, β-catenin, and β-actin levels were detected by Western blot analysis. The results shown are representative of three independent experiments. (D) Quantitation of C. The results presented in this study are the representative images of three independent experiments (n = 3) and are expressed as means ± SE; *P < .05; **P < .01. (E, F) Immunofluorescence staining using anti-Met, p-c-Met, anti-Stat3, and β-catenin antibodies followed by an Alexa Fluor 594-tagged secondary antibody was performed in shEGFR-transfected U251 and 5310 cells compared to the SV controls. Panels were photographed at 20x. Red indicates the expression levels of the respective proteins, whereas DAPI is used to stain the nucleus. Scale bars, 200 µm.