Fig 4. Lapatinib and Th1 cytokines suppress HER-family RTK expression and phosphorylation status.

SK-BR-3 and MDA-MB-468 cells were treated with either Th1 cytokines (TNF-α plus IFN-γ; 20 and 12.5ng/ml respectively), 200nM Lapatinib, Th1 cytokines plus Lapatinib, or left untreated (control). A) After 72 hours incubation the cells were harvested, extracted in presence protease and phosphatase inhibitors, and 30μg/well total proteins separated on a 4–15% gradient SDS-PAGE gel prior to electrotransfer onto nitrocellulose membranes. Blots were then probed with anti-HER2, anti-phospho-HER2, anti-HER3 and anti-phospho-HER3 antibodies, and bands detected using chemiluminescence. B) After 24 hours, MDA-MB-468 cells treated as in (A) were supplied with caspase antagonist Z-DEVD-F. After 72 hours total culture cells were harvested and stained with EGFR antibody conjugated to PE/Dazzel 594 and analyzed for EGFR expression via flow cytometry.