Figure 4.
The generation of trastuzumab resistance induced changes in markers phosphorylation patterns in BCCLs. Trastuzumab-sensitive and -resistant BT-474, SK-BR-3, AU-565, and EFM-192A cell lines were compared for their degree of signaling nodes phosphorylation at baseline and after trastuzumab-treatment conditions. Representative images of WB assays are depicted, with annotation of the intensity values as obtained by densitometric analysis of the bands (the intensity values of the phosphorylated proteins were normalized to their corresponding total protein bands; non-phosphorylated bands were normalized to GAPDH). Initial seeding was carried out in the absence of treatment, and subsequently trastuzumab was added in a time-dependent manner. Trastuzumab induced changes in phosphorylation dependent on the exposure time. For all experiments, 1.5 × 106 cells/plate were seeded (except for BT-474, in which 1.7 × 106 cells/plate were cultured). Trastuzumab was added at 15 µg/ml after 24 h of culture and maintained for either 48 h or 72 h more, when cells were then collected for protein extraction. All conditions were therefore processed simultaneously.