Journal: Molecular carcinogenesis

Article Title: Soluble-E-Cadherin Activates HER and IAP Family Members in HER2+ and TNBC Human Breast Cancers

doi: 10.1002/mc.22048

Figure Lengend Snippet: Enhanced cleavage of the sEcad ectodomain fragment in breast carcinogenesis. (A) Western blot analysis and representative immunoblots of endogenous sEcad levels in normal human mammary tissues (N) from reductive mammoplasty and resected human HER2+ breast specimens (T). Protein levels were normalized to β-actin and quantified using NIH Scion Image. **P < 0.01 versus N. (B) Western blot analysis and representative immunoblots of endogenous sEcad levels in resected human TNBC specimens (T) with adjacent normals (N). Protein levels were normalized to G3PDH and quantified using NIH Scion Image. *P < 0.05 versus N. (C) Analysis of sEcad levels in the conditioned medium of normal mammary cells (MCF-10A), SKBR3, MDA-MB-231 and MCF-7 cells (in the presence or absence of 10 µM of GM6001) by ELISA after normalization for cell number. Insert: Immunoblot of intact E-cadherin and sEcad in cell lystes from MCF-7 cells treated with or without GM6001. ***P < 0.001 versus normal mammary cells MCF-10A; ###P < 0.001 versus MCF-7 without GM6001. (D) Western blot analysis of endogenous sEcad levels in resected normal mouse mammary tissues (WT) and MMTV-PyMT breast tumors (MMTV) relative to G3PDH. (E) Concentration of sEcad in the serum and urine of WT and MMTV-PyMT mice as measured by ELISA. Urinary levels of sEcad were normalized to creatinine. Data are presented as Mean ± SEM. *P < 0.05, **P < 0.01 versus corresponding WT controls.

Article Snippet: HER2+ human breast cancer specimens were obtained from the NCI Cooperative Human Tissue Network (CHTN) and from Proteogenex (Culver City, CA).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Molecular carcinogenesis

Article Title: Soluble-E-Cadherin Activates HER and IAP Family Members in HER2+ and TNBC Human Breast Cancers

doi: 10.1002/mc.22048

Figure Lengend Snippet: sEcad associates with the HER family of receptors in human and mouse breast tumors and cells. (A) HER1, HER2, and HER3 were immunoprecipitated (IP) from human ductal adenocarcinoma lysates and precipitates were immunoblotted (IB) using anti-E-cadherin extracellular domain, anti-HER1, anti-HER2, and anti-HER3 antibodies. sEcad-HER association was confirmed by reverse immunoprecipitation, whereby HER1, HER2, and HER3 were detected by immunoblot analysis following immunoprecipitation of E-cadherin using an E-cadherin ectodomain-specific antibody. (B) HER1 was IP from human triple negative breast carcinoma (TNBC) specimens and precipitates were immunoblotted using anti-HER1 and anti-E-cadherin extracellular domain antibodies. (C) HER1, HER2, and HER3 were IP from resected MMTV-PyMT mouse tumors and precipitates were immunoblotted using anti-E-cadherin extracellular domain, anti-HER1, anti-HER2, and anti-HER3 specific antibodies. (D) Lysates from MCF-7, SKBR3, and MDA-MB-231 cells in the presence or absence of 10 µg/ml of recombinant E-cadherin ectodomain-Fc chimeric protein (rhEcad/Fc; sEcad) fused with a C-terminal His Tag were IP with anti-HER1, anti- HER2, anti-HER3, or anti-HER4 specific antibodies, followed by immunoblotting with an anti-His Tag antibody.

Article Snippet: HER2+ human breast cancer specimens were obtained from the NCI Cooperative Human Tissue Network (CHTN) and from Proteogenex (Culver City, CA).

Techniques: Immunoprecipitation, Western Blot, Recombinant