Western Blot Analysis of Lapatinib-Mediated Inhibition of the Epidermal Growth Factor Receptor 2 (HER2) Pathway in Breast Cancer Cells.

ABSTRACT

Western blotting is an excellent technique to investigate aberrations and/or therapy-induced changes in signaling proteins in cancer. Using an in vitro system, we prepared whole cell lysates from HER2-overexpressing breast cancer cell lines, treated or not with the tyrosine kinase inhibitor, lapatinib, in the presence and absence of IFN-γ. Here we describe the protocol whereby proteins in the lysates were separated by SDS-PAGE, electrophoretically transferred to nitrocellulose membranes followed by an enzyme-linked immunoassay and chemiluminescence to reveal the relevant phosphorylated and dephosphorylated proteins. Herein, Western blot analysis confirmed lapatinib dephosphorylated HER2 and downstream signaling proteins and IFN-γ induced phosphorylation of STAT1.