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Figure 6 | Breast Cancer Research

Figure 6

From: Polyclonal HER2-specific antibodies induced by vaccination mediate receptor internalization and degradation in tumor cells

Figure 6

HER2 ubiquitination, degradation, and fragmentation induced by vaccine-induced anti-HER2 antibodies. (A) Vaccine-induced anti-HER2 antibodies (HER2-VIA) stimulation led to HER2 ubiquitination. SK-BR-3 cells expressing ubiquitin-Myc were pretreated with MG132 for 2 hours and then treated with HER2-VIA for the indicated time. The ubiquitinated HER2 was detected by anti-Myc antibody. (B) HER2-VIA stimulation leads to HER2 ubiquitination. SK-BR-3 cells were pretreated with MG132 for 2 hours before HER2-VIA stimulation. Cell lysates were immunoprecipitated with anti-HER2 29D8 antibody. The endogenous ubiquitinated HER2 was detected by the anti-ubiquitin antibody and the total HER2 was visualized by anti-HER2 3B5 antibody. (C), (D) HER2-VIA stimulation causes HER2 degradation: (C) SK-BR-3 and (D) HCC1569 cells were stimulated with HER2-VIA, LacZ-VIA, or trastuzumab for the indicated time, and an equal amount of cell lysates was subjected to western blot analysis. Expression levels of HER2 and β-actin were detected by corresponding antibodies. (E) HER2-VIA stimulation produces HER2 fragmentation. SK-BR-3 cells were treated with HER2-VIA for 6 hours in the absence or presence of prior MG132 treatment for 2 hours. Full-length and truncated HER2 are detected by anti-HER2 3B5 antibody. β-actin serves as loading control. (F) HER2-VIA stimulation produces tyrosine phosphorylation of the 130 kDa HER2 C-terminal fragment. SK-BR-3 cells were incubated with HER2-VIA for 6 hours after pretreatment with lapatinib, MG132, or lapatinib plus MG132 for 2 hours. Full-length and truncated HER2 are detected by anti-HER2 3B5 antibody, which recognizes the C-terminus (top panel). Phosphorylated full-length and truncated HER2 were recognized by tyrosine site-specific phospho-antibodies for phosphorylated tyrosine 877, 1,221/1,222 and 1,248 (middle three panels). IB, immunoblot.

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