Reduced signaling of HER2 following patient's serum treatment. (A) Imaging of endogenous HER2 internalization by the human HER2-specific antibodies and vaccine-induced anti-HER2 antibodies (HER2-VIA). All human serum samples were purified to deplete lapatinib prior to using in the internalization assay. SK-BR-3 cells were allowed to grow for 24 hours and were treated for 1 hour with different murine or human antibodies as indicated above each image. Following treatment, the cells were fixed and stained to visualize the cellular distribution of HER2 using HER2 antibody 29D8 as described in Materials and methods. Confocal images of cells that (a) were left untreated, or were treated with (b) control GFP-VIA, (c) HER2-VIA, (d, e) serum (week 0 and week 10) from Patient 8, (f, g) serum (week 0 and week 12) from Patient 2, and (h, i) serum (week 0 and week 12) from Patient 4. (B) Effect of human HER2-specific antibodies on Her2 tyrosine 877 phosphorylation. SK-BR-3 cells were stimulated for 9 hours with indicated sera from either patients or mice. Protein samples were immunoblotted with anti-Her2PY877 antibodies (upper panel), anti-Her2 antibodies (middle panel), or anti-β-actin antibodies as a loading control (lower panel). n = 3. IB, immunoblot; NS, non-stimulated.