EGFR phosphorylation levels after EGF stimulation, sialidase NEU3 overexpression and gefitinib treatment.

(A) Representative Western-blot analyses performed on a normal lung cell line (HSAEC1) and NSCLC cell lines (HCC4006 and H1734) transfected either with the empty vector (mock) or pcDNA3.1-HsNEU3. Cells were treated for 3 h with 1 μM gefitinib, followed by the addition of EGF (20 ng/mL) for 15 min. Protein extracts were separated on a 10% SDS-PAGE and probed with anti-EGFR, anti-P-EGFR antibodies. GAPDH was used as a loading control. The experiments were performed in triplicate. (B)–(C)–(D) Densitometric analysis was performed with Scion Image Software. Values are expressed by comparing the data obtained after EGF stimulation with those obtained in the absence of EGF (B); by comparing the data obtained after transfection with NEU3 with those obtained after transfection with the empty vector (mock) (C); by comparing the data obtained after gefitinib treatment with those obtained without gefitinib administration (D). Statistical analyses were performed using Student’s t-test. Values are presented as means ± standard error (SE). *p<0.05, **p<0.01, and ***p<0.001 (Student’s t-test).