Fig 1: Immunofluorescent staining of breast cancer cell lines using Oslo-2 mAb. Among different breast cancer cell lines, Oslo-2 mAb was only reactive to the p95HER2-T47D cell line and did not show any reactivity to HER2+ SK-BR-3 and MDA-MB-468, while Cytopla-HER2 stained positively p95HER2-T47D and HER2+ SK-BR-3 and MDA-MB-468 cell lines. Neither Oslo-2 mAb nor Cytoplasm-HER2 was reactive to HER2- T47D. Oslo-2 mAb signals were overlapped with Cytopla-HER2 signals, confirming the specific binding of Oslo-2 mAb to p95HER2.
Fig 2: Oslo-2 mAb epitope mapping. (a) Serial overlapping peptides covering the p95HER2 extracellular domain, with 11 aa overlap between consecutive peptides. (b) Signal intensity representing binding of Oslo-2 mAb to respective peptides, showing that Oslo-2 mAb was reactive only to peptide 1, containing “MPIW”. The assay was performed in triplicates. (c) 3D structure of extracellular part of full-length HER2, with p95HER2 region highlighted. (d) Peptides 1–20: Truncated peptides covering 633–622 in full-length HER2, truncated from N-terminal. Peptides 21–39: Two amino acid alanine substitutions in peptides covering 633–622 in full-length HER2. (e) Signal intensity representing binding of Oslo-2 mAb to respective peptides (duplicates). Truncated peptide data shows that sequence PIW is crucial for binding. Alanine substitution data reveals that KFPDEE is also required. (f) 3D structure of full-length HER2 and p95HER2, depicting that the Oslo-2 mAb binding epitope, PIWKFPDEE, is continuous and hidden in full-length HER2. The sequence and color coding for the p95HER2 are shown on the right.
Fig 3: Expression of NRG1 (a) and ErbB2 (b) is demonstrated by Western blotting normalized to GAPDH. Panels above the graph are representative Western blot images from different groups. *P < 0.05 vs. control rats (two-way ANOVA). DC: diabetic control rats; DT: diabetic and trained; HC: health and control; HT: health and trained. Sol: soleus; EDL: extensor digitorum longus. Five rats in each group were utilized for this analysis. The analysis was performed in triplicate.
Fig 4: HA@MOF@GSK-J1 affects a JMJD3-mediated gene network. (A) Heat-map of gene expression in CR SKOV-3 cells after different treatments (B) KEGG analysis of the biological pathways of the genes in regulated CR SKOV-3 cells following treatment with HA@MOF@GSK-J1 compared to treatment with MOF@GSK-J1 treated. (C) Histograms showing the RNA fold-induction of different treatments. qPCR analysis of CR SKOV-3 cells was performed using the indicated primers after different treatments (Supplementary Table S1). Amplification of GAPDH transcripts was used to normalize the loading of each RNA sample. (D) ChIP-qPCR analysis of H3K27me3 mark of the HER2, and MYCN promoters in CR SKOV-3 cells treated with different formulations. (E) Western blot analysis of HER2 in CR SKOV-3 cells after different treatments. All results are expressed as fold-inductions (means) from three independent experiments. The bars indicate SD (*p < 0.05, Student’s t-tests).
Fig 5: p95HER2 poly and monoclonal hybridoma selection. (a) The top nine polyclonal hybridomas from the initial iQue screening were tested by flow cytometry for binding to breast cancer target cells expressing 611-p95HER2 (p95HER2--T47D) or full-length HER2 (SK-BR-3), and HER2 negative controls including wild type T-47D and SUP-T1 (blue: secondary antibody alone and red: secondary + primary antibodies). pClone 2 had the strongest p95HER2-reactivity, followed by pClones 1, 3 and 8. pClones 2 and 8 were weekly reactive to SK-BR-3 (HER2+), while pClone 1 and 3 had no detectable reactivity against SK-BR-3. None of the pClones were reactive to the HER2 negative target cells (T-47D or SUP-T1). (b) Flow cytometry screening of supernatants from monoclonal hybridomas (mClones) generated from pClones 1, 2 and 3. mClone 1 stained p95HER-T47D, and not SK-BR-3. mClone 2 stained both p95HER-T47D and SK-BR-3, while mClone 3 stained neither. (c) The same batch of p95HER-T47D cells was stained for immunofluorescence with supernatants from pClone 1 and mClone 1. These results indicate that the reactivity of Clone 1 to p95HER2 was increased from the poly- to monoclonal level.
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