Mapping of the mAb 1479 epitope.

<p>(A) A schematic illustration of different His-tagged extracellular domain constructs generated to map mAb 1479 binding to ErbB4. (B) Western analysis of conditioned media from COS-7 cell transfectants expressing the different His-tagged subdomains of ErbB4 extracellular domain using mAb 1479 or anti-pentaHis as the primary antibody. (C, D) Crystal structure of the sErbB4:Fab 1479 complex. (C) A ribbon diagram of the sErbB4:1479 Fab complex is shown. sErbB4 is colored blue (subdomain I), green (subdomain II), yellow (subdomain III), and red (subdomain IV). The 1479 heavy chain is colored cyan and the light chain palecyan. The side chains of sErbB4 residues containing atoms within 4 Å of 1479 atoms are shown as stick models. (D) An expanded view of the sErbB4:Fab 1479 interface with sErbB4 residues containing atoms within 4 Å of Fab 1479 shown as stick models. The orientation is similar to that shown in panel C.</p

Association of ErbB4 expression and localization with clinical variables.

<p><i>P</i> values <0.05 are shown in bold.</p

Immunohistochemical and ELISA analysis of ErbB4 expression in breast cancer tissues and matched serum samples.

<p>(A, B) Representative examples of immunohistochemical analysis of breast cancer sections with an antibody (HFR-1) recognizing the intracellular domain of ErbB4 demonstrate strong cytoplasmic immunoreactivity in the absence of nuclear staining (A), or strong cytoplasmic and strong nuclear immunoreactivity (B). (C) ELISA analysis of ErbB4 ectodomain levels in serum samples from 30 healthy individuals (white dots) and 238 breast cancer patients (black dots). (D) ROC analysis of the value of serum ErbB4 ectodomain concentration in differentiating between normal and cancer. AUC for ErbB4 concentration in serum was 0.76. At a cut-off of 40 ng/mL of ErbB4 ectodomain, the specificity of the assay was 100% and the sensitivity 43%. All samples with ErbB4 level below the detection limit of the assay (51% of cancer samples, 47% of control samples) were excluded from the analysis.</p

Effect of estrogen on ErbB4 shedding.

<p>(A) Western analysis of MCF-7 cells treated with or without 10 nM estradiol or 100 ng/mL PMA for 24 hours. The amount of shed ErbB4 ectodomain (100 kD) was detected with mAb 1479 under non-reducing conditions from culture medium and full-length ErbB4 (180 kD) with anti-ErbB4 (E-200) from cell lysates. Anti-actin was used as a loading control. The experiment was repeated four times with similar results. (B) TACE activity assay of MCF-7 cells treated for 30 min with or without 10 nM estradiol or 100 ng/mL PMA. After the treatments, TACE substrate peptide that becomes fluorescent upon cleavage was applied to the cells at time point 0. *, <i>P</i><0.05 compared to control at the time point.</p