Journal: PLoS ONE

Article Title: The Human Splice Variant Δ16HER2 Induces Rapid Tumor Onset in a Reporter Transgenic Mouse

doi: 10.1371/journal.pone.0018727

Figure Lengend Snippet: (A) Schematic representation of the MMTV-driven human Δ16HER2-LUC transgene, with the MMTV LTR promoter (pMMTV, red), the human Δ16HER2 cDNA (green), the IRES (internal ribosome entry site, yellow), the luciferase cDNA (LUC, purple), and the termination signal from the SV40 (Poly A, blue). Relevant restriction sites are indicated. (B) Graphic representation of murine chromosome 5 divided into A–G regions and numbered subregions, showing that the transgene integrates in region E-1. (C, D) An F2 female transgenic mouse with primary breast tumors (arrows) just before tumor removal. (E) Bioluminescence analysis of a 28-week-old tumor-bearing Δ16HER2-LUC transgenic mouse (founder female), using the in VivoVision Systems, Xenogen. (F and G) Immunohistochemical detection of HER2 and luciferase, respectively, showing strong and uniform expression of both proteins in sections of a mammary tumor, while the normal duct on the right appears negative. Magnification: ×400.

Article Snippet: Membranes were probed with mouse monoclonal antibodies (MAbs) Ab3 (1 µg/ml; Calbiochem, Darmstadt, Germany), anti-Src (1∶1000; Cell Signaling Technology, Beverly, MA), anti-phospho-Stat3 (1∶1000; Santa Cruz Biotechnology, Santa Cruz, CA), anti-actin AC-40 (0.4 mg/ml; Sigma), PoAb anti-phospho-HER2 p-Neu (tyr 1248; 0.5 mg/ml, Santa Cruz Biotechnology), anti-phospho-p44/42 MAPK (Erk1/2; 1∶2000; Cell Signaling Technology), anti-p44/42 MAPK (Erk1/2; 1∶1.000; Cell Signaling Technology), anti-phospho-Akt (Ser 473; 1∶1000; Cell Signaling Technology), anti-Akt (1∶1000; Cell Signaling Technology), anti-phospho-Src (Tyr 416; 1∶1000; Cell Signaling Technology), anti-Stat3 (1∶1000; Santa Cruz Biotechnology) and anti-E-cadherin (#4065, 1∶1000; Cell Signaling Technology).

Techniques: Luciferase, Transgenic Assay, Immunohistochemical staining, Expressing

Journal: PLoS ONE

Article Title: The Human Splice Variant Δ16HER2 Induces Rapid Tumor Onset in a Reporter Transgenic Mouse

doi: 10.1371/journal.pone.0018727

Figure Lengend Snippet: (A) Protein extracts from 4 different Δ16HER2 tumor samples (lanes 1–4) and breast cancer cell lines BT474 (lane 5) and ZR-75.1 (lane 6) were separated by 4–12% gradient SDS-PAGE under non-reducing conditions and probed, respectively, with MAb Ab3 (HER2 M and D) and PoAb anti-phospho-HER2 p-neu (p-HER2 M and D). (B) The same protein extracts (lanes 1–6) were analyzed under reducing conditions for activation of the following HER2 downstream oncogenic signaling pathways: MAPK, AKT, Src and STAT3. Actin was used to normalize protein loading. (C) Proteins from the pool of whole Δ16HER2 tumor extracts (lanes 1, subpanels a and b) and purified plasma membrane extracts (lanes 2, subpanels a and b) were separated by 4–12% gradient SDS-PAGE under non-reducing conditions and probed with MAb Ab3 (a) and PoAb antiphospho-HER2 p-neu (b). E-cadherin was used as a plasma membrane marker.

Article Snippet: Membranes were probed with mouse monoclonal antibodies (MAbs) Ab3 (1 µg/ml; Calbiochem, Darmstadt, Germany), anti-Src (1∶1000; Cell Signaling Technology, Beverly, MA), anti-phospho-Stat3 (1∶1000; Santa Cruz Biotechnology, Santa Cruz, CA), anti-actin AC-40 (0.4 mg/ml; Sigma), PoAb anti-phospho-HER2 p-Neu (tyr 1248; 0.5 mg/ml, Santa Cruz Biotechnology), anti-phospho-p44/42 MAPK (Erk1/2; 1∶2000; Cell Signaling Technology), anti-p44/42 MAPK (Erk1/2; 1∶1.000; Cell Signaling Technology), anti-phospho-Akt (Ser 473; 1∶1000; Cell Signaling Technology), anti-Akt (1∶1000; Cell Signaling Technology), anti-phospho-Src (Tyr 416; 1∶1000; Cell Signaling Technology), anti-Stat3 (1∶1000; Santa Cruz Biotechnology) and anti-E-cadherin (#4065, 1∶1000; Cell Signaling Technology).

Techniques: SDS Page, Activation Assay, Purification, Marker

Journal: PLoS ONE

Article Title: The Human Splice Variant Δ16HER2 Induces Rapid Tumor Onset in a Reporter Transgenic Mouse

doi: 10.1371/journal.pone.0018727

Figure Lengend Snippet: (A) Bioluminescence analysis of an F2 female transgenic mouse at 14 weeks of age and (B) whole-mount analysis of the inguinal mammary glands; arrows indicate non-palpable tumors. (C) Hematoxylin-eosin and immunohistochemical staining for HER2 (D) and PCNA (E) of non-palpable mammary tumors at 14 weeks. Magnification: B, ×6; C–E, ×400.

Article Snippet: Membranes were probed with mouse monoclonal antibodies (MAbs) Ab3 (1 µg/ml; Calbiochem, Darmstadt, Germany), anti-Src (1∶1000; Cell Signaling Technology, Beverly, MA), anti-phospho-Stat3 (1∶1000; Santa Cruz Biotechnology, Santa Cruz, CA), anti-actin AC-40 (0.4 mg/ml; Sigma), PoAb anti-phospho-HER2 p-Neu (tyr 1248; 0.5 mg/ml, Santa Cruz Biotechnology), anti-phospho-p44/42 MAPK (Erk1/2; 1∶2000; Cell Signaling Technology), anti-p44/42 MAPK (Erk1/2; 1∶1.000; Cell Signaling Technology), anti-phospho-Akt (Ser 473; 1∶1000; Cell Signaling Technology), anti-Akt (1∶1000; Cell Signaling Technology), anti-phospho-Src (Tyr 416; 1∶1000; Cell Signaling Technology), anti-Stat3 (1∶1000; Santa Cruz Biotechnology) and anti-E-cadherin (#4065, 1∶1000; Cell Signaling Technology).

Techniques: Transgenic Assay, Immunohistochemical staining, Staining