Figure 4

Pharmacological blockade of EGFR does not inhibit IGF-1 induced increases in LIP expression. MCF10A cells were serum-starved for 24 hrs, pretreated for 30-60 min with inhibitors, and stimulated with ligands for 10 min or 16 hours. A). Western blot analysis demonstrating that pretreatment of cells with the EGFR inhibitor, AG1478, does not prevent IGF-1R induced LIP expression at 16 hrs, but does antagonize EGF induced LIP expression at doses of 0.1, 1, and 5 μM as compared to control cells stimulated with EGF but without AG1478 treatment (0). The functionality of AG1478 was confirmed by Western blot using a p-EGFR antibody (lower panel). B). LIP/LAPs ratio in MCF10A cells stimulated with IGF-1 and treated with AG1478 as in (A) was quantitated using an Odyssey infrared imaging system. No significant decrease of IGF-1R induced LIP expression or LIP/LAPs ratio was observed with AG1478 treatment at 16 hours as determined via a two-tailed T-test (n = 3). C). Western blot demonstrating that increasing doses of AG1478 (0.1-1 μM) can inhibit EGF induced phosphorylation of Erk1/2 and Akt, and the IGF induced phosphorylation of Erk1/2, but AG1478 has no effect on IGF-1R activated phosphorylation of Akt. Total Erk1/2 and Akt served as loading controls. D). Western blot analysis demonstrates that treatment with mAb528 only blocks the EGF induced LIP expression, p-Akt, and p-Erk1/2. There is no obvious effect on IGF-1R induced LIP expression, p-Akt, or p-Erk1/2.