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anti phospho her2 erbb2  (Cell Signaling Technology Inc)


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    Structured Review

    Cell Signaling Technology Inc anti phospho her2 erbb2
    Anti Phospho Her2 Erbb2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 312 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/her2+rabbit+mab/Phospho-HER2%2FErbB2+(Tyr1221%2F1222)+Rabbit+mAb/pmc13041752-3-0-3
    Average 96 stars, based on 312 article reviews
    anti phospho her2 erbb2 - by Bioz Stars, 2026-10
    96/100 stars

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    Related Articles

    Incubation:

    Article Title: Pyrotinib promotes the antitumor effect of T-DM1 by increasing drug endocytosis in HER2-positive breast cancer.
    Article Snippet: .. After being treated with pyrotinib and lapatinib, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and incubated with a blocking solution and HER2 Rabbit mAb (Cell Signaling Technology) at 4 °C overnight. .. Subsequently, the cells were incubated with Goat Anti-Rabbit IgG (H + L) Alexa Fluor 488 (Shanghai Abways Biotechnology Co., Ltd., Shanghai, China) for 1 h at room temperature.

    Article Title: New Class of Hsp90 C-Terminal Domain Inhibitors with Anti-tumor Properties against Triple-Negative Breast Cancer.
    Article Snippet: Electrophoresis was performed at 80 V for 15 min and then at 130 V for 60 min, followed by transfer to a PVDF membrane using the iBlot3 Dry Blotting System (Thermo Fisher Scientific, Waltham, MA, USA). .. Nonspecific binding sites were blocked with 5% BSA for 1 h at room temperature, followed by for 18 h incubation at 4 °C with primary antibodies against Hsp90 Rabbit mAb (1:1000), Hsp70 Mouse mAb (1:1000), cRAF Rabbit mAb (1:1000), GAPDH Rabbit mAb (1:2500), AKT Rabbit mAb (1:1000), phospho-AKT rabbit mAb (1:1000), phospho-MEK rabbit mAb (1:1000), MEK rabbit mAb (1:1000), phospho-ERK mouse mAb (1:1000), ERK mouse mAb (1:1000), CDK4 rabbit mAb (1:1000), ERα rabbit mAb (1:1000), Her2 rabbit mAb (1:1000), β-tubulin rabbit mAb (1:5000), and β-actin mouse mAb (1:5000) (all antibodies from Cell Signaling, Danvers, MA, USA). ..

    Article Title: New Class of Hsp90 C-Terminal Domain Inhibitors with Anti-tumor Properties against Triple-Negative Breast Cancer
    Article Snippet: Electrophoresis was performed at 80 V for 15 min and then at 130 V for 60 min, followed by transfer to a PVDF membrane using the iBlot3 Dry Blotting System (Thermo Fisher Scientific, Waltham, MA, USA). .. Nonspecific binding sites were blocked with 5% BSA for 1 h at room temperature, followed by for 18 h incubation at 4 °C with primary antibodies against Hsp90 Rabbit mAb (1:1000), Hsp70 Mouse mAb (1:1000), cRAF Rabbit mAb (1:1000), GAPDH Rabbit mAb (1:2500), AKT Rabbit mAb (1:1000), phospho-AKT rabbit mAb (1:1000), phospho-MEK rabbit mAb (1:1000), MEK rabbit mAb (1:1000), phospho-ERK mouse mAb (1:1000), ERK mouse mAb (1:1000), CDK4 rabbit mAb (1:1000), ERα rabbit mAb (1:1000), Her2 rabbit mAb (1:1000), β-tubulin rabbit mAb (1:5000), and β-actin mouse mAb (1:5000) (all antibodies from Cell Signaling, Danvers, MA, USA). ..

    Article Title: Pyrotinib promotes the antitumor effect of T-DM1 by increasing drug endocytosis in HER2-positive breast cancer
    Article Snippet: .. After being treated with pyrotinib and lapatinib, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and incubated with a blocking solution and HER2 Rabbit mAb (Cell Signaling Technology) at 4 °C overnight. .. Subsequently, the cells were incubated with Goat Anti-Rabbit IgG (H + L) Alexa Fluor 488 (Shanghai Abways Biotechnology Co., Ltd., Shanghai, China) for 1 h at room temperature.

    Blocking Assay:

    Article Title: Pyrotinib promotes the antitumor effect of T-DM1 by increasing drug endocytosis in HER2-positive breast cancer.
    Article Snippet: .. After being treated with pyrotinib and lapatinib, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and incubated with a blocking solution and HER2 Rabbit mAb (Cell Signaling Technology) at 4 °C overnight. .. Subsequently, the cells were incubated with Goat Anti-Rabbit IgG (H + L) Alexa Fluor 488 (Shanghai Abways Biotechnology Co., Ltd., Shanghai, China) for 1 h at room temperature.

    Article Title: Pyrotinib promotes the antitumor effect of T-DM1 by increasing drug endocytosis in HER2-positive breast cancer
    Article Snippet: .. After being treated with pyrotinib and lapatinib, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and incubated with a blocking solution and HER2 Rabbit mAb (Cell Signaling Technology) at 4 °C overnight. .. Subsequently, the cells were incubated with Goat Anti-Rabbit IgG (H + L) Alexa Fluor 488 (Shanghai Abways Biotechnology Co., Ltd., Shanghai, China) for 1 h at room temperature.

    Binding Assay:

    Article Title: New Class of Hsp90 C-Terminal Domain Inhibitors with Anti-tumor Properties against Triple-Negative Breast Cancer.
    Article Snippet: Electrophoresis was performed at 80 V for 15 min and then at 130 V for 60 min, followed by transfer to a PVDF membrane using the iBlot3 Dry Blotting System (Thermo Fisher Scientific, Waltham, MA, USA). .. Nonspecific binding sites were blocked with 5% BSA for 1 h at room temperature, followed by for 18 h incubation at 4 °C with primary antibodies against Hsp90 Rabbit mAb (1:1000), Hsp70 Mouse mAb (1:1000), cRAF Rabbit mAb (1:1000), GAPDH Rabbit mAb (1:2500), AKT Rabbit mAb (1:1000), phospho-AKT rabbit mAb (1:1000), phospho-MEK rabbit mAb (1:1000), MEK rabbit mAb (1:1000), phospho-ERK mouse mAb (1:1000), ERK mouse mAb (1:1000), CDK4 rabbit mAb (1:1000), ERα rabbit mAb (1:1000), Her2 rabbit mAb (1:1000), β-tubulin rabbit mAb (1:5000), and β-actin mouse mAb (1:5000) (all antibodies from Cell Signaling, Danvers, MA, USA). ..

    Article Title: New Class of Hsp90 C-Terminal Domain Inhibitors with Anti-tumor Properties against Triple-Negative Breast Cancer
    Article Snippet: Electrophoresis was performed at 80 V for 15 min and then at 130 V for 60 min, followed by transfer to a PVDF membrane using the iBlot3 Dry Blotting System (Thermo Fisher Scientific, Waltham, MA, USA). .. Nonspecific binding sites were blocked with 5% BSA for 1 h at room temperature, followed by for 18 h incubation at 4 °C with primary antibodies against Hsp90 Rabbit mAb (1:1000), Hsp70 Mouse mAb (1:1000), cRAF Rabbit mAb (1:1000), GAPDH Rabbit mAb (1:2500), AKT Rabbit mAb (1:1000), phospho-AKT rabbit mAb (1:1000), phospho-MEK rabbit mAb (1:1000), MEK rabbit mAb (1:1000), phospho-ERK mouse mAb (1:1000), ERK mouse mAb (1:1000), CDK4 rabbit mAb (1:1000), ERα rabbit mAb (1:1000), Her2 rabbit mAb (1:1000), β-tubulin rabbit mAb (1:5000), and β-actin mouse mAb (1:5000) (all antibodies from Cell Signaling, Danvers, MA, USA). ..

    other:




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    A TTNT with T-DXd by <t>HER2</t> status of the disease; B TTNT with T-DXd by clinical subtype of the disease; C TTNT with T-DXd depending on changes in HER2 status (low vs. 0) between the primary tumor and last metastatic biopsy; D TTNT with T-DXd depending on changes in HER2 status (low vs. 0) between the first and most recent metastatic biopsy; E OS with T-DXd by HER2 status of the disease; F OS with T-DXd by clinical subtype of the disease. T-DXd, trastuzumab deruxtecan; TTNT, time to next treatment; HER2, human epidermal growth factor receptor 2; OS, overall survival; HR, hormone receptor; TNBC, triple-negative breast cancer.
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    Image Search Results


    EBA downregulates HER2, p95HER2, HER3 and AKT expression. (A) Immunoblot analysis of HER2, p95HER2 and p-HER2 (Y1221/1222) in JIMT-1 cells treated with EBA for 48 h. (B) Immunoblot analysis of HER3, p-HER3 (Y1289), AKT and p-AKT following treatment with EBA (48 h) in JIMT-1 cells. (C) Immunoblot analysis of HER2, HER3 and EGFR following IP with anti-HER2 antibody in JIMT-1 cells treated with EBA. In silico molecular docking of EBA with the crystal structure of HER2-KD. (D) Surface map of lipophilic and hydrophilic properties at the ATP-binding site of HER2-KD (red, hydrophobic; blue, hydrophilic). (E) 2D interaction diagram showing intermolecular interactions between EBA and HER2-KD. Key amino acid residues within the binding pocket are shown. (F) Predicted binding pose of EBA (purple stick model) within the tyrosine kinase domain of HER2 (blue ribbon). (G) 293T cells were treated with DMSO or EBA for 1 h at 37°C, followed by heating for 3 min. Soluble fractions were collected following centrifugation and analyzed by immunoblotting using an anti-HER2 antibody. EBA, ebastine; p-, phosphorylated; IP, immunoprecipitation; IB, immunoblotting; KD, kinase domain PCB, protein complex binding; TM, transmembrane; a.a., amino acid.

    Journal: International Journal of Molecular Medicine

    Article Title: Ebastine targets HER2/HER3 signaling and cancer stem cell traits to overcome trastuzumab resistance in HER2-positive breast cancer

    doi: 10.3892/ijmm.2026.5751

    Figure Lengend Snippet: EBA downregulates HER2, p95HER2, HER3 and AKT expression. (A) Immunoblot analysis of HER2, p95HER2 and p-HER2 (Y1221/1222) in JIMT-1 cells treated with EBA for 48 h. (B) Immunoblot analysis of HER3, p-HER3 (Y1289), AKT and p-AKT following treatment with EBA (48 h) in JIMT-1 cells. (C) Immunoblot analysis of HER2, HER3 and EGFR following IP with anti-HER2 antibody in JIMT-1 cells treated with EBA. In silico molecular docking of EBA with the crystal structure of HER2-KD. (D) Surface map of lipophilic and hydrophilic properties at the ATP-binding site of HER2-KD (red, hydrophobic; blue, hydrophilic). (E) 2D interaction diagram showing intermolecular interactions between EBA and HER2-KD. Key amino acid residues within the binding pocket are shown. (F) Predicted binding pose of EBA (purple stick model) within the tyrosine kinase domain of HER2 (blue ribbon). (G) 293T cells were treated with DMSO or EBA for 1 h at 37°C, followed by heating for 3 min. Soluble fractions were collected following centrifugation and analyzed by immunoblotting using an anti-HER2 antibody. EBA, ebastine; p-, phosphorylated; IP, immunoprecipitation; IB, immunoblotting; KD, kinase domain PCB, protein complex binding; TM, transmembrane; a.a., amino acid.

    Article Snippet: Primary antibodies were as follows: Ki-67 (cat. no. ab16667), CD31 (cat. no. ab28364), ALDH1A1 (Abcam; cat. no. ab52492), Bcl-2 (Abcam; cat. no. ab692) and CD44 (all Abcam; cat. no. ab254530); HER2 (Cell Signaling Technology, Inc.; cat. no. 2165), HER3 (Cell Signaling Technology, Inc.; cat. no. 12708), phosphorylated (p-)HER2 (Y1221/1222; Cell Signaling Technology, Inc.; cat. no. 2243), p-HER3 (Y1289; Cell Signaling Technology, Inc.; cat. no. 2842), Akt (Cell Signaling Technology, Inc.; cat. no. 9272), p-Akt (S473; Cell Signaling Technology, Inc.; cat. no. 4060), PARP (Cell Signaling Technology, Inc.; cat. no. 9542), cleaved PARP (Cell Signaling Technology, Inc.; cat. no. 5625), caspase-3 (Cell Signaling Technology, Inc.; cat. no. 7148), -7 (Cell Signaling Technology, Inc.; cat. no. 12827) and -8 (Cell Signaling Technology, Inc.; cat. no. 4790), cleaved caspase-3 (Cell Signaling Technology, Inc.; cat. no. 9664), -7 (Cell Signaling Technology, Inc.; cat. no. 8438) and -8 (Cell Signaling Technology, Inc.; cat. no. 9496), Bax (Cell Signaling Technology, Inc.; cat. no. 2772) and vimentin (Cell Signaling Technology, Inc.; cat. no. 5741); anti-intracellular domain (ICD) HER2 clone 4B5 (Ventana Medical Systems; cat. no. 790-4493) and GAPDH (Invitrogen; Thermo Fisher Scientific, Inc.; cat. no. MA5-15738).

    Techniques: Expressing, Western Blot, In Silico, Binding Assay, Centrifugation, Immunoprecipitation

    EBA impairs cancer stem cell-like properties. (A) BT474 and SKBR3 cells were treated with EBA for 48 h, and ALDH1 activity was assessed by flow cytometry using the Aldefluor assay. DEAB was used to define the baseline of Aldefluor-positive fluorescence. (B) BT474 cells (5x10 4 cells/ml) were plated in ultra-low attachment dishes and cultured in the presence or absence of EBA for 5 days. The number and volume of mammospheres were measured by microscopy. (C) Overall survival of patients with breast cancer stratified by the co-expression of ALDH1A1 and CD44. (D) Spearman correlation analysis of ALDH1A1 and CD44 mRNA levels in patients with HER2-positive breast cancer from The Cancer Genome Atlas cohort (n=76). Kaplan-Meier survival analyses of patients with HER2-overexpressing breast cancer stratified by (E) ALDH1A1 and (F) CD44 expression. Patients were divided into high- and low-expression groups based on the median gene expression. Statistical significance was determined using the log-rank test. (G) JIMT-1 cells were treated with EBA (3 μ M) for 48 h and the CD44 high /CD24 low cell populations were identified by flow cytometry. (H) JIMT-1 cells (1.5x10 4 cells/ml) were cultured under serum-free suspension conditions in the presence of EBA (3 μ M) for 8 days. Mammosphere number and volumes were quantified. ** P<0.01 and **** P<0.0001 vs. vehicle-treated control (0 μ M EBA). EBA, ebastine; ALDH, aldehyde dehydrogenase; DEAB, diethylaminobenzaldehyde; CTL, control; ISO, isotype.

    Journal: International Journal of Molecular Medicine

    Article Title: Ebastine targets HER2/HER3 signaling and cancer stem cell traits to overcome trastuzumab resistance in HER2-positive breast cancer

    doi: 10.3892/ijmm.2026.5751

    Figure Lengend Snippet: EBA impairs cancer stem cell-like properties. (A) BT474 and SKBR3 cells were treated with EBA for 48 h, and ALDH1 activity was assessed by flow cytometry using the Aldefluor assay. DEAB was used to define the baseline of Aldefluor-positive fluorescence. (B) BT474 cells (5x10 4 cells/ml) were plated in ultra-low attachment dishes and cultured in the presence or absence of EBA for 5 days. The number and volume of mammospheres were measured by microscopy. (C) Overall survival of patients with breast cancer stratified by the co-expression of ALDH1A1 and CD44. (D) Spearman correlation analysis of ALDH1A1 and CD44 mRNA levels in patients with HER2-positive breast cancer from The Cancer Genome Atlas cohort (n=76). Kaplan-Meier survival analyses of patients with HER2-overexpressing breast cancer stratified by (E) ALDH1A1 and (F) CD44 expression. Patients were divided into high- and low-expression groups based on the median gene expression. Statistical significance was determined using the log-rank test. (G) JIMT-1 cells were treated with EBA (3 μ M) for 48 h and the CD44 high /CD24 low cell populations were identified by flow cytometry. (H) JIMT-1 cells (1.5x10 4 cells/ml) were cultured under serum-free suspension conditions in the presence of EBA (3 μ M) for 8 days. Mammosphere number and volumes were quantified. ** P<0.01 and **** P<0.0001 vs. vehicle-treated control (0 μ M EBA). EBA, ebastine; ALDH, aldehyde dehydrogenase; DEAB, diethylaminobenzaldehyde; CTL, control; ISO, isotype.

    Article Snippet: Primary antibodies were as follows: Ki-67 (cat. no. ab16667), CD31 (cat. no. ab28364), ALDH1A1 (Abcam; cat. no. ab52492), Bcl-2 (Abcam; cat. no. ab692) and CD44 (all Abcam; cat. no. ab254530); HER2 (Cell Signaling Technology, Inc.; cat. no. 2165), HER3 (Cell Signaling Technology, Inc.; cat. no. 12708), phosphorylated (p-)HER2 (Y1221/1222; Cell Signaling Technology, Inc.; cat. no. 2243), p-HER3 (Y1289; Cell Signaling Technology, Inc.; cat. no. 2842), Akt (Cell Signaling Technology, Inc.; cat. no. 9272), p-Akt (S473; Cell Signaling Technology, Inc.; cat. no. 4060), PARP (Cell Signaling Technology, Inc.; cat. no. 9542), cleaved PARP (Cell Signaling Technology, Inc.; cat. no. 5625), caspase-3 (Cell Signaling Technology, Inc.; cat. no. 7148), -7 (Cell Signaling Technology, Inc.; cat. no. 12827) and -8 (Cell Signaling Technology, Inc.; cat. no. 4790), cleaved caspase-3 (Cell Signaling Technology, Inc.; cat. no. 9664), -7 (Cell Signaling Technology, Inc.; cat. no. 8438) and -8 (Cell Signaling Technology, Inc.; cat. no. 9496), Bax (Cell Signaling Technology, Inc.; cat. no. 2772) and vimentin (Cell Signaling Technology, Inc.; cat. no. 5741); anti-intracellular domain (ICD) HER2 clone 4B5 (Ventana Medical Systems; cat. no. 790-4493) and GAPDH (Invitrogen; Thermo Fisher Scientific, Inc.; cat. no. MA5-15738).

    Techniques: Activity Assay, Flow Cytometry, Fluorescence, Cell Culture, Microscopy, Expressing, Gene Expression, Suspension, Control

    EBA downregulates HER2, ICD-HER2, HER3, ALDH1A1, CD44 and vimentin in JIMT-1 xenograft tumors. Immunofluorescence staining of JIMT-1 xenograft tumor tissue for (A) full-length HER2 (green), (B) ICD-HER2 (green) and (C) HER3. Immunohistochemical analysis of (D) ALDH1A1 (green) and (E) CD44 (red) in tumor tissue. (F) Tumor sections were immunostained for vimentin (red). Magnification, x500. Fluorescence intensities were quantified. Serum biochemical analysis for (G) liver and (H) kidney function in EBA-treated or CTL mice (n=5). Serum levels of ALT, AST, TBL, BUN and creatinine were assessed. *** P<0.001, **** P<0.0001. ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen; EBA, ebastine; ICD, intracellular domain; ALDH, aldehyde dehydrogenase; CTL, control; NS, not significant.

    Journal: International Journal of Molecular Medicine

    Article Title: Ebastine targets HER2/HER3 signaling and cancer stem cell traits to overcome trastuzumab resistance in HER2-positive breast cancer

    doi: 10.3892/ijmm.2026.5751

    Figure Lengend Snippet: EBA downregulates HER2, ICD-HER2, HER3, ALDH1A1, CD44 and vimentin in JIMT-1 xenograft tumors. Immunofluorescence staining of JIMT-1 xenograft tumor tissue for (A) full-length HER2 (green), (B) ICD-HER2 (green) and (C) HER3. Immunohistochemical analysis of (D) ALDH1A1 (green) and (E) CD44 (red) in tumor tissue. (F) Tumor sections were immunostained for vimentin (red). Magnification, x500. Fluorescence intensities were quantified. Serum biochemical analysis for (G) liver and (H) kidney function in EBA-treated or CTL mice (n=5). Serum levels of ALT, AST, TBL, BUN and creatinine were assessed. *** P<0.001, **** P<0.0001. ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen; EBA, ebastine; ICD, intracellular domain; ALDH, aldehyde dehydrogenase; CTL, control; NS, not significant.

    Article Snippet: Primary antibodies were as follows: Ki-67 (cat. no. ab16667), CD31 (cat. no. ab28364), ALDH1A1 (Abcam; cat. no. ab52492), Bcl-2 (Abcam; cat. no. ab692) and CD44 (all Abcam; cat. no. ab254530); HER2 (Cell Signaling Technology, Inc.; cat. no. 2165), HER3 (Cell Signaling Technology, Inc.; cat. no. 12708), phosphorylated (p-)HER2 (Y1221/1222; Cell Signaling Technology, Inc.; cat. no. 2243), p-HER3 (Y1289; Cell Signaling Technology, Inc.; cat. no. 2842), Akt (Cell Signaling Technology, Inc.; cat. no. 9272), p-Akt (S473; Cell Signaling Technology, Inc.; cat. no. 4060), PARP (Cell Signaling Technology, Inc.; cat. no. 9542), cleaved PARP (Cell Signaling Technology, Inc.; cat. no. 5625), caspase-3 (Cell Signaling Technology, Inc.; cat. no. 7148), -7 (Cell Signaling Technology, Inc.; cat. no. 12827) and -8 (Cell Signaling Technology, Inc.; cat. no. 4790), cleaved caspase-3 (Cell Signaling Technology, Inc.; cat. no. 9664), -7 (Cell Signaling Technology, Inc.; cat. no. 8438) and -8 (Cell Signaling Technology, Inc.; cat. no. 9496), Bax (Cell Signaling Technology, Inc.; cat. no. 2772) and vimentin (Cell Signaling Technology, Inc.; cat. no. 5741); anti-intracellular domain (ICD) HER2 clone 4B5 (Ventana Medical Systems; cat. no. 790-4493) and GAPDH (Invitrogen; Thermo Fisher Scientific, Inc.; cat. no. MA5-15738).

    Techniques: Immunofluorescence, Staining, Immunohistochemical staining, Fluorescence, Control

    EBA downregulates HER2, p95HER2, HER3 and AKT expression. (A) Immunoblot analysis of HER2, p95HER2 and p-HER2 (Y1221/1222) in JIMT-1 cells treated with EBA for 48 h. (B) Immunoblot analysis of HER3, p-HER3 (Y1289), AKT and p-AKT following treatment with EBA (48 h) in JIMT-1 cells. (C) Immunoblot analysis of HER2, HER3 and EGFR following IP with anti-HER2 antibody in JIMT-1 cells treated with EBA. In silico molecular docking of EBA with the crystal structure of HER2-KD. (D) Surface map of lipophilic and hydrophilic properties at the ATP-binding site of HER2-KD (red, hydrophobic; blue, hydrophilic). (E) 2D interaction diagram showing intermolecular interactions between EBA and HER2-KD. Key amino acid residues within the binding pocket are shown. (F) Predicted binding pose of EBA (purple stick model) within the tyrosine kinase domain of HER2 (blue ribbon). (G) 293T cells were treated with DMSO or EBA for 1 h at 37°C, followed by heating for 3 min. Soluble fractions were collected following centrifugation and analyzed by immunoblotting using an anti-HER2 antibody. EBA, ebastine; p-, phosphorylated; IP, immunoprecipitation; IB, immunoblotting; KD, kinase domain PCB, protein complex binding; TM, transmembrane; a.a., amino acid.

    Journal: International Journal of Molecular Medicine

    Article Title: Ebastine targets HER2/HER3 signaling and cancer stem cell traits to overcome trastuzumab resistance in HER2-positive breast cancer

    doi: 10.3892/ijmm.2026.5751

    Figure Lengend Snippet: EBA downregulates HER2, p95HER2, HER3 and AKT expression. (A) Immunoblot analysis of HER2, p95HER2 and p-HER2 (Y1221/1222) in JIMT-1 cells treated with EBA for 48 h. (B) Immunoblot analysis of HER3, p-HER3 (Y1289), AKT and p-AKT following treatment with EBA (48 h) in JIMT-1 cells. (C) Immunoblot analysis of HER2, HER3 and EGFR following IP with anti-HER2 antibody in JIMT-1 cells treated with EBA. In silico molecular docking of EBA with the crystal structure of HER2-KD. (D) Surface map of lipophilic and hydrophilic properties at the ATP-binding site of HER2-KD (red, hydrophobic; blue, hydrophilic). (E) 2D interaction diagram showing intermolecular interactions between EBA and HER2-KD. Key amino acid residues within the binding pocket are shown. (F) Predicted binding pose of EBA (purple stick model) within the tyrosine kinase domain of HER2 (blue ribbon). (G) 293T cells were treated with DMSO or EBA for 1 h at 37°C, followed by heating for 3 min. Soluble fractions were collected following centrifugation and analyzed by immunoblotting using an anti-HER2 antibody. EBA, ebastine; p-, phosphorylated; IP, immunoprecipitation; IB, immunoblotting; KD, kinase domain PCB, protein complex binding; TM, transmembrane; a.a., amino acid.

    Article Snippet: Primary antibodies were as follows: Ki-67 (cat. no. ab16667), CD31 (cat. no. ab28364), ALDH1A1 (Abcam; cat. no. ab52492), Bcl-2 (Abcam; cat. no. ab692) and CD44 (all Abcam; cat. no. ab254530); HER2 (Cell Signaling Technology, Inc.; cat. no. 2165), HER3 (Cell Signaling Technology, Inc.; cat. no. 12708), phosphorylated (p-)HER2 (Y1221/1222; Cell Signaling Technology, Inc.; cat. no. 2243), p-HER3 (Y1289; Cell Signaling Technology, Inc.; cat. no. 2842), Akt (Cell Signaling Technology, Inc.; cat. no. 9272), p-Akt (S473; Cell Signaling Technology, Inc.; cat. no. 4060), PARP (Cell Signaling Technology, Inc.; cat. no. 9542), cleaved PARP (Cell Signaling Technology, Inc.; cat. no. 5625), caspase-3 (Cell Signaling Technology, Inc.; cat. no. 7148), -7 (Cell Signaling Technology, Inc.; cat. no. 12827) and -8 (Cell Signaling Technology, Inc.; cat. no. 4790), cleaved caspase-3 (Cell Signaling Technology, Inc.; cat. no. 9664), -7 (Cell Signaling Technology, Inc.; cat. no. 8438) and -8 (Cell Signaling Technology, Inc.; cat. no. 9496), Bax (Cell Signaling Technology, Inc.; cat. no. 2772) and vimentin (Cell Signaling Technology, Inc.; cat. no. 5741); anti-intracellular domain (ICD) HER2 clone 4B5 (Ventana Medical Systems; cat. no. 790-4493) and GAPDH (Invitrogen; Thermo Fisher Scientific, Inc.; cat. no. MA5-15738).

    Techniques: Expressing, Western Blot, In Silico, Binding Assay, Centrifugation, Immunoprecipitation

    EBA downregulates HER2, ICD-HER2, HER3, ALDH1A1, CD44 and vimentin in JIMT-1 xenograft tumors. Immunofluorescence staining of JIMT-1 xenograft tumor tissue for (A) full-length HER2 (green), (B) ICD-HER2 (green) and (C) HER3. Immunohistochemical analysis of (D) ALDH1A1 (green) and (E) CD44 (red) in tumor tissue. (F) Tumor sections were immunostained for vimentin (red). Magnification, x500. Fluorescence intensities were quantified. Serum biochemical analysis for (G) liver and (H) kidney function in EBA-treated or CTL mice (n=5). Serum levels of ALT, AST, TBL, BUN and creatinine were assessed. *** P<0.001, **** P<0.0001. ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen; EBA, ebastine; ICD, intracellular domain; ALDH, aldehyde dehydrogenase; CTL, control; NS, not significant.

    Journal: International Journal of Molecular Medicine

    Article Title: Ebastine targets HER2/HER3 signaling and cancer stem cell traits to overcome trastuzumab resistance in HER2-positive breast cancer

    doi: 10.3892/ijmm.2026.5751

    Figure Lengend Snippet: EBA downregulates HER2, ICD-HER2, HER3, ALDH1A1, CD44 and vimentin in JIMT-1 xenograft tumors. Immunofluorescence staining of JIMT-1 xenograft tumor tissue for (A) full-length HER2 (green), (B) ICD-HER2 (green) and (C) HER3. Immunohistochemical analysis of (D) ALDH1A1 (green) and (E) CD44 (red) in tumor tissue. (F) Tumor sections were immunostained for vimentin (red). Magnification, x500. Fluorescence intensities were quantified. Serum biochemical analysis for (G) liver and (H) kidney function in EBA-treated or CTL mice (n=5). Serum levels of ALT, AST, TBL, BUN and creatinine were assessed. *** P<0.001, **** P<0.0001. ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen; EBA, ebastine; ICD, intracellular domain; ALDH, aldehyde dehydrogenase; CTL, control; NS, not significant.

    Article Snippet: Primary antibodies were as follows: Ki-67 (cat. no. ab16667), CD31 (cat. no. ab28364), ALDH1A1 (Abcam; cat. no. ab52492), Bcl-2 (Abcam; cat. no. ab692) and CD44 (all Abcam; cat. no. ab254530); HER2 (Cell Signaling Technology, Inc.; cat. no. 2165), HER3 (Cell Signaling Technology, Inc.; cat. no. 12708), phosphorylated (p-)HER2 (Y1221/1222; Cell Signaling Technology, Inc.; cat. no. 2243), p-HER3 (Y1289; Cell Signaling Technology, Inc.; cat. no. 2842), Akt (Cell Signaling Technology, Inc.; cat. no. 9272), p-Akt (S473; Cell Signaling Technology, Inc.; cat. no. 4060), PARP (Cell Signaling Technology, Inc.; cat. no. 9542), cleaved PARP (Cell Signaling Technology, Inc.; cat. no. 5625), caspase-3 (Cell Signaling Technology, Inc.; cat. no. 7148), -7 (Cell Signaling Technology, Inc.; cat. no. 12827) and -8 (Cell Signaling Technology, Inc.; cat. no. 4790), cleaved caspase-3 (Cell Signaling Technology, Inc.; cat. no. 9664), -7 (Cell Signaling Technology, Inc.; cat. no. 8438) and -8 (Cell Signaling Technology, Inc.; cat. no. 9496), Bax (Cell Signaling Technology, Inc.; cat. no. 2772) and vimentin (Cell Signaling Technology, Inc.; cat. no. 5741); anti-intracellular domain (ICD) HER2 clone 4B5 (Ventana Medical Systems; cat. no. 790-4493) and GAPDH (Invitrogen; Thermo Fisher Scientific, Inc.; cat. no. MA5-15738).

    Techniques: Immunofluorescence, Staining, Immunohistochemical staining, Fluorescence, Control

    EBA downregulates HER2, p95HER2, HER3 and AKT expression. (A) Immunoblot analysis of HER2, p95HER2 and p-HER2 (Y1221/1222) in JIMT-1 cells treated with EBA for 48 h. (B) Immunoblot analysis of HER3, p-HER3 (Y1289), AKT and p-AKT following treatment with EBA (48 h) in JIMT-1 cells. (C) Immunoblot analysis of HER2, HER3 and EGFR following IP with anti-HER2 antibody in JIMT-1 cells treated with EBA. In silico molecular docking of EBA with the crystal structure of HER2-KD. (D) Surface map of lipophilic and hydrophilic properties at the ATP-binding site of HER2-KD (red, hydrophobic; blue, hydrophilic). (E) 2D interaction diagram showing intermolecular interactions between EBA and HER2-KD. Key amino acid residues within the binding pocket are shown. (F) Predicted binding pose of EBA (purple stick model) within the tyrosine kinase domain of HER2 (blue ribbon). (G) 293T cells were treated with DMSO or EBA for 1 h at 37°C, followed by heating for 3 min. Soluble fractions were collected following centrifugation and analyzed by immunoblotting using an anti-HER2 antibody. EBA, ebastine; p-, phosphorylated; IP, immunoprecipitation; IB, immunoblotting; KD, kinase domain PCB, protein complex binding; TM, transmembrane; a.a., amino acid.

    Journal: International Journal of Molecular Medicine

    Article Title: Ebastine targets HER2/HER3 signaling and cancer stem cell traits to overcome trastuzumab resistance in HER2-positive breast cancer

    doi: 10.3892/ijmm.2026.5751

    Figure Lengend Snippet: EBA downregulates HER2, p95HER2, HER3 and AKT expression. (A) Immunoblot analysis of HER2, p95HER2 and p-HER2 (Y1221/1222) in JIMT-1 cells treated with EBA for 48 h. (B) Immunoblot analysis of HER3, p-HER3 (Y1289), AKT and p-AKT following treatment with EBA (48 h) in JIMT-1 cells. (C) Immunoblot analysis of HER2, HER3 and EGFR following IP with anti-HER2 antibody in JIMT-1 cells treated with EBA. In silico molecular docking of EBA with the crystal structure of HER2-KD. (D) Surface map of lipophilic and hydrophilic properties at the ATP-binding site of HER2-KD (red, hydrophobic; blue, hydrophilic). (E) 2D interaction diagram showing intermolecular interactions between EBA and HER2-KD. Key amino acid residues within the binding pocket are shown. (F) Predicted binding pose of EBA (purple stick model) within the tyrosine kinase domain of HER2 (blue ribbon). (G) 293T cells were treated with DMSO or EBA for 1 h at 37°C, followed by heating for 3 min. Soluble fractions were collected following centrifugation and analyzed by immunoblotting using an anti-HER2 antibody. EBA, ebastine; p-, phosphorylated; IP, immunoprecipitation; IB, immunoblotting; KD, kinase domain PCB, protein complex binding; TM, transmembrane; a.a., amino acid.

    Article Snippet: Primary antibodies were as follows: Ki-67 (cat. no. ab16667), CD31 (cat. no. ab28364), ALDH1A1 (Abcam; cat. no. ab52492), Bcl-2 (Abcam; cat. no. ab692) and CD44 (all Abcam; cat. no. ab254530); HER2 (Cell Signaling Technology, Inc.; cat. no. 2165), HER3 (Cell Signaling Technology, Inc.; cat. no. 12708), phosphorylated (p-)HER2 (Y1221/1222; Cell Signaling Technology, Inc.; cat. no. 2243), p-HER3 (Y1289; Cell Signaling Technology, Inc.; cat. no. 2842), Akt (Cell Signaling Technology, Inc.; cat. no. 9272), p-Akt (S473; Cell Signaling Technology, Inc.; cat. no. 4060), PARP (Cell Signaling Technology, Inc.; cat. no. 9542), cleaved PARP (Cell Signaling Technology, Inc.; cat. no. 5625), caspase-3 (Cell Signaling Technology, Inc.; cat. no. 7148), -7 (Cell Signaling Technology, Inc.; cat. no. 12827) and -8 (Cell Signaling Technology, Inc.; cat. no. 4790), cleaved caspase-3 (Cell Signaling Technology, Inc.; cat. no. 9664), -7 (Cell Signaling Technology, Inc.; cat. no. 8438) and -8 (Cell Signaling Technology, Inc.; cat. no. 9496), Bax (Cell Signaling Technology, Inc.; cat. no. 2772) and vimentin (Cell Signaling Technology, Inc.; cat. no. 5741); anti-intracellular domain (ICD) HER2 clone 4B5 (Ventana Medical Systems; cat. no. 790-4493) and GAPDH (Invitrogen; Thermo Fisher Scientific, Inc.; cat. no. MA5-15738).

    Techniques: Expressing, Western Blot, In Silico, Binding Assay, Centrifugation, Immunoprecipitation

    EBA downregulates HER2, ICD-HER2, HER3, ALDH1A1, CD44 and vimentin in JIMT-1 xenograft tumors. Immunofluorescence staining of JIMT-1 xenograft tumor tissue for (A) full-length HER2 (green), (B) ICD-HER2 (green) and (C) HER3. Immunohistochemical analysis of (D) ALDH1A1 (green) and (E) CD44 (red) in tumor tissue. (F) Tumor sections were immunostained for vimentin (red). Magnification, x500. Fluorescence intensities were quantified. Serum biochemical analysis for (G) liver and (H) kidney function in EBA-treated or CTL mice (n=5). Serum levels of ALT, AST, TBL, BUN and creatinine were assessed. *** P<0.001, **** P<0.0001. ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen; EBA, ebastine; ICD, intracellular domain; ALDH, aldehyde dehydrogenase; CTL, control; NS, not significant.

    Journal: International Journal of Molecular Medicine

    Article Title: Ebastine targets HER2/HER3 signaling and cancer stem cell traits to overcome trastuzumab resistance in HER2-positive breast cancer

    doi: 10.3892/ijmm.2026.5751

    Figure Lengend Snippet: EBA downregulates HER2, ICD-HER2, HER3, ALDH1A1, CD44 and vimentin in JIMT-1 xenograft tumors. Immunofluorescence staining of JIMT-1 xenograft tumor tissue for (A) full-length HER2 (green), (B) ICD-HER2 (green) and (C) HER3. Immunohistochemical analysis of (D) ALDH1A1 (green) and (E) CD44 (red) in tumor tissue. (F) Tumor sections were immunostained for vimentin (red). Magnification, x500. Fluorescence intensities were quantified. Serum biochemical analysis for (G) liver and (H) kidney function in EBA-treated or CTL mice (n=5). Serum levels of ALT, AST, TBL, BUN and creatinine were assessed. *** P<0.001, **** P<0.0001. ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen; EBA, ebastine; ICD, intracellular domain; ALDH, aldehyde dehydrogenase; CTL, control; NS, not significant.

    Article Snippet: Primary antibodies were as follows: Ki-67 (cat. no. ab16667), CD31 (cat. no. ab28364), ALDH1A1 (Abcam; cat. no. ab52492), Bcl-2 (Abcam; cat. no. ab692) and CD44 (all Abcam; cat. no. ab254530); HER2 (Cell Signaling Technology, Inc.; cat. no. 2165), HER3 (Cell Signaling Technology, Inc.; cat. no. 12708), phosphorylated (p-)HER2 (Y1221/1222; Cell Signaling Technology, Inc.; cat. no. 2243), p-HER3 (Y1289; Cell Signaling Technology, Inc.; cat. no. 2842), Akt (Cell Signaling Technology, Inc.; cat. no. 9272), p-Akt (S473; Cell Signaling Technology, Inc.; cat. no. 4060), PARP (Cell Signaling Technology, Inc.; cat. no. 9542), cleaved PARP (Cell Signaling Technology, Inc.; cat. no. 5625), caspase-3 (Cell Signaling Technology, Inc.; cat. no. 7148), -7 (Cell Signaling Technology, Inc.; cat. no. 12827) and -8 (Cell Signaling Technology, Inc.; cat. no. 4790), cleaved caspase-3 (Cell Signaling Technology, Inc.; cat. no. 9664), -7 (Cell Signaling Technology, Inc.; cat. no. 8438) and -8 (Cell Signaling Technology, Inc.; cat. no. 9496), Bax (Cell Signaling Technology, Inc.; cat. no. 2772) and vimentin (Cell Signaling Technology, Inc.; cat. no. 5741); anti-intracellular domain (ICD) HER2 clone 4B5 (Ventana Medical Systems; cat. no. 790-4493) and GAPDH (Invitrogen; Thermo Fisher Scientific, Inc.; cat. no. MA5-15738).

    Techniques: Immunofluorescence, Staining, Immunohistochemical staining, Fluorescence, Control

    EBA downregulates HER2, p95HER2, HER3 and AKT expression. (A) Immunoblot analysis of HER2, p95HER2 and p-HER2 (Y1221/1222) in JIMT-1 cells treated with EBA for 48 h. (B) Immunoblot analysis of HER3, p-HER3 (Y1289), AKT and p-AKT following treatment with EBA (48 h) in JIMT-1 cells. (C) Immunoblot analysis of HER2, HER3 and EGFR following IP with anti-HER2 antibody in JIMT-1 cells treated with EBA. In silico molecular docking of EBA with the crystal structure of HER2-KD. (D) Surface map of lipophilic and hydrophilic properties at the ATP-binding site of HER2-KD (red, hydrophobic; blue, hydrophilic). (E) 2D interaction diagram showing intermolecular interactions between EBA and HER2-KD. Key amino acid residues within the binding pocket are shown. (F) Predicted binding pose of EBA (purple stick model) within the tyrosine kinase domain of HER2 (blue ribbon). (G) 293T cells were treated with DMSO or EBA for 1 h at 37°C, followed by heating for 3 min. Soluble fractions were collected following centrifugation and analyzed by immunoblotting using an anti-HER2 antibody. EBA, ebastine; p-, phosphorylated; IP, immunoprecipitation; IB, immunoblotting; KD, kinase domain PCB, protein complex binding; TM, transmembrane; a.a., amino acid.

    Journal: International Journal of Molecular Medicine

    Article Title: Ebastine targets HER2/HER3 signaling and cancer stem cell traits to overcome trastuzumab resistance in HER2-positive breast cancer

    doi: 10.3892/ijmm.2026.5751

    Figure Lengend Snippet: EBA downregulates HER2, p95HER2, HER3 and AKT expression. (A) Immunoblot analysis of HER2, p95HER2 and p-HER2 (Y1221/1222) in JIMT-1 cells treated with EBA for 48 h. (B) Immunoblot analysis of HER3, p-HER3 (Y1289), AKT and p-AKT following treatment with EBA (48 h) in JIMT-1 cells. (C) Immunoblot analysis of HER2, HER3 and EGFR following IP with anti-HER2 antibody in JIMT-1 cells treated with EBA. In silico molecular docking of EBA with the crystal structure of HER2-KD. (D) Surface map of lipophilic and hydrophilic properties at the ATP-binding site of HER2-KD (red, hydrophobic; blue, hydrophilic). (E) 2D interaction diagram showing intermolecular interactions between EBA and HER2-KD. Key amino acid residues within the binding pocket are shown. (F) Predicted binding pose of EBA (purple stick model) within the tyrosine kinase domain of HER2 (blue ribbon). (G) 293T cells were treated with DMSO or EBA for 1 h at 37°C, followed by heating for 3 min. Soluble fractions were collected following centrifugation and analyzed by immunoblotting using an anti-HER2 antibody. EBA, ebastine; p-, phosphorylated; IP, immunoprecipitation; IB, immunoblotting; KD, kinase domain PCB, protein complex binding; TM, transmembrane; a.a., amino acid.

    Article Snippet: Primary antibodies were as follows: Ki-67 (cat. no. ab16667), CD31 (cat. no. ab28364), ALDH1A1 (Abcam; cat. no. ab52492), Bcl-2 (Abcam; cat. no. ab692) and CD44 (all Abcam; cat. no. ab254530); HER2 (Cell Signaling Technology, Inc.; cat. no. 2165), HER3 (Cell Signaling Technology, Inc.; cat. no. 12708), phosphorylated (p-)HER2 (Y1221/1222; Cell Signaling Technology, Inc.; cat. no. 2243), p-HER3 (Y1289; Cell Signaling Technology, Inc.; cat. no. 2842), Akt (Cell Signaling Technology, Inc.; cat. no. 9272), p-Akt (S473; Cell Signaling Technology, Inc.; cat. no. 4060), PARP (Cell Signaling Technology, Inc.; cat. no. 9542), cleaved PARP (Cell Signaling Technology, Inc.; cat. no. 5625), caspase-3 (Cell Signaling Technology, Inc.; cat. no. 7148), -7 (Cell Signaling Technology, Inc.; cat. no. 12827) and -8 (Cell Signaling Technology, Inc.; cat. no. 4790), cleaved caspase-3 (Cell Signaling Technology, Inc.; cat. no. 9664), -7 (Cell Signaling Technology, Inc.; cat. no. 8438) and -8 (Cell Signaling Technology, Inc.; cat. no. 9496), Bax (Cell Signaling Technology, Inc.; cat. no. 2772) and vimentin (Cell Signaling Technology, Inc.; cat. no. 5741); anti-intracellular domain (ICD) HER2 clone 4B5 (Ventana Medical Systems; cat. no. 790-4493) and GAPDH (Invitrogen; Thermo Fisher Scientific, Inc.; cat. no. MA5-15738).

    Techniques: Expressing, Western Blot, In Silico, Binding Assay, Centrifugation, Immunoprecipitation

    EBA impairs cancer stem cell-like properties. (A) BT474 and SKBR3 cells were treated with EBA for 48 h, and ALDH1 activity was assessed by flow cytometry using the Aldefluor assay. DEAB was used to define the baseline of Aldefluor-positive fluorescence. (B) BT474 cells (5x10 4 cells/ml) were plated in ultra-low attachment dishes and cultured in the presence or absence of EBA for 5 days. The number and volume of mammospheres were measured by microscopy. (C) Overall survival of patients with breast cancer stratified by the co-expression of ALDH1A1 and CD44. (D) Spearman correlation analysis of ALDH1A1 and CD44 mRNA levels in patients with HER2-positive breast cancer from The Cancer Genome Atlas cohort (n=76). Kaplan-Meier survival analyses of patients with HER2-overexpressing breast cancer stratified by (E) ALDH1A1 and (F) CD44 expression. Patients were divided into high- and low-expression groups based on the median gene expression. Statistical significance was determined using the log-rank test. (G) JIMT-1 cells were treated with EBA (3 μ M) for 48 h and the CD44 high /CD24 low cell populations were identified by flow cytometry. (H) JIMT-1 cells (1.5x10 4 cells/ml) were cultured under serum-free suspension conditions in the presence of EBA (3 μ M) for 8 days. Mammosphere number and volumes were quantified. ** P<0.01 and **** P<0.0001 vs. vehicle-treated control (0 μ M EBA). EBA, ebastine; ALDH, aldehyde dehydrogenase; DEAB, diethylaminobenzaldehyde; CTL, control; ISO, isotype.

    Journal: International Journal of Molecular Medicine

    Article Title: Ebastine targets HER2/HER3 signaling and cancer stem cell traits to overcome trastuzumab resistance in HER2-positive breast cancer

    doi: 10.3892/ijmm.2026.5751

    Figure Lengend Snippet: EBA impairs cancer stem cell-like properties. (A) BT474 and SKBR3 cells were treated with EBA for 48 h, and ALDH1 activity was assessed by flow cytometry using the Aldefluor assay. DEAB was used to define the baseline of Aldefluor-positive fluorescence. (B) BT474 cells (5x10 4 cells/ml) were plated in ultra-low attachment dishes and cultured in the presence or absence of EBA for 5 days. The number and volume of mammospheres were measured by microscopy. (C) Overall survival of patients with breast cancer stratified by the co-expression of ALDH1A1 and CD44. (D) Spearman correlation analysis of ALDH1A1 and CD44 mRNA levels in patients with HER2-positive breast cancer from The Cancer Genome Atlas cohort (n=76). Kaplan-Meier survival analyses of patients with HER2-overexpressing breast cancer stratified by (E) ALDH1A1 and (F) CD44 expression. Patients were divided into high- and low-expression groups based on the median gene expression. Statistical significance was determined using the log-rank test. (G) JIMT-1 cells were treated with EBA (3 μ M) for 48 h and the CD44 high /CD24 low cell populations were identified by flow cytometry. (H) JIMT-1 cells (1.5x10 4 cells/ml) were cultured under serum-free suspension conditions in the presence of EBA (3 μ M) for 8 days. Mammosphere number and volumes were quantified. ** P<0.01 and **** P<0.0001 vs. vehicle-treated control (0 μ M EBA). EBA, ebastine; ALDH, aldehyde dehydrogenase; DEAB, diethylaminobenzaldehyde; CTL, control; ISO, isotype.

    Article Snippet: Primary antibodies were as follows: Ki-67 (cat. no. ab16667), CD31 (cat. no. ab28364), ALDH1A1 (Abcam; cat. no. ab52492), Bcl-2 (Abcam; cat. no. ab692) and CD44 (all Abcam; cat. no. ab254530); HER2 (Cell Signaling Technology, Inc.; cat. no. 2165), HER3 (Cell Signaling Technology, Inc.; cat. no. 12708), phosphorylated (p-)HER2 (Y1221/1222; Cell Signaling Technology, Inc.; cat. no. 2243), p-HER3 (Y1289; Cell Signaling Technology, Inc.; cat. no. 2842), Akt (Cell Signaling Technology, Inc.; cat. no. 9272), p-Akt (S473; Cell Signaling Technology, Inc.; cat. no. 4060), PARP (Cell Signaling Technology, Inc.; cat. no. 9542), cleaved PARP (Cell Signaling Technology, Inc.; cat. no. 5625), caspase-3 (Cell Signaling Technology, Inc.; cat. no. 7148), -7 (Cell Signaling Technology, Inc.; cat. no. 12827) and -8 (Cell Signaling Technology, Inc.; cat. no. 4790), cleaved caspase-3 (Cell Signaling Technology, Inc.; cat. no. 9664), -7 (Cell Signaling Technology, Inc.; cat. no. 8438) and -8 (Cell Signaling Technology, Inc.; cat. no. 9496), Bax (Cell Signaling Technology, Inc.; cat. no. 2772) and vimentin (Cell Signaling Technology, Inc.; cat. no. 5741); anti-intracellular domain (ICD) HER2 clone 4B5 (Ventana Medical Systems; cat. no. 790-4493) and GAPDH (Invitrogen; Thermo Fisher Scientific, Inc.; cat. no. MA5-15738).

    Techniques: Activity Assay, Flow Cytometry, Fluorescence, Cell Culture, Microscopy, Expressing, Gene Expression, Suspension, Control

    EBA downregulates HER2, ICD-HER2, HER3, ALDH1A1, CD44 and vimentin in JIMT-1 xenograft tumors. Immunofluorescence staining of JIMT-1 xenograft tumor tissue for (A) full-length HER2 (green), (B) ICD-HER2 (green) and (C) HER3. Immunohistochemical analysis of (D) ALDH1A1 (green) and (E) CD44 (red) in tumor tissue. (F) Tumor sections were immunostained for vimentin (red). Magnification, x500. Fluorescence intensities were quantified. Serum biochemical analysis for (G) liver and (H) kidney function in EBA-treated or CTL mice (n=5). Serum levels of ALT, AST, TBL, BUN and creatinine were assessed. *** P<0.001, **** P<0.0001. ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen; EBA, ebastine; ICD, intracellular domain; ALDH, aldehyde dehydrogenase; CTL, control; NS, not significant.

    Journal: International Journal of Molecular Medicine

    Article Title: Ebastine targets HER2/HER3 signaling and cancer stem cell traits to overcome trastuzumab resistance in HER2-positive breast cancer

    doi: 10.3892/ijmm.2026.5751

    Figure Lengend Snippet: EBA downregulates HER2, ICD-HER2, HER3, ALDH1A1, CD44 and vimentin in JIMT-1 xenograft tumors. Immunofluorescence staining of JIMT-1 xenograft tumor tissue for (A) full-length HER2 (green), (B) ICD-HER2 (green) and (C) HER3. Immunohistochemical analysis of (D) ALDH1A1 (green) and (E) CD44 (red) in tumor tissue. (F) Tumor sections were immunostained for vimentin (red). Magnification, x500. Fluorescence intensities were quantified. Serum biochemical analysis for (G) liver and (H) kidney function in EBA-treated or CTL mice (n=5). Serum levels of ALT, AST, TBL, BUN and creatinine were assessed. *** P<0.001, **** P<0.0001. ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen; EBA, ebastine; ICD, intracellular domain; ALDH, aldehyde dehydrogenase; CTL, control; NS, not significant.

    Article Snippet: Primary antibodies were as follows: Ki-67 (cat. no. ab16667), CD31 (cat. no. ab28364), ALDH1A1 (Abcam; cat. no. ab52492), Bcl-2 (Abcam; cat. no. ab692) and CD44 (all Abcam; cat. no. ab254530); HER2 (Cell Signaling Technology, Inc.; cat. no. 2165), HER3 (Cell Signaling Technology, Inc.; cat. no. 12708), phosphorylated (p-)HER2 (Y1221/1222; Cell Signaling Technology, Inc.; cat. no. 2243), p-HER3 (Y1289; Cell Signaling Technology, Inc.; cat. no. 2842), Akt (Cell Signaling Technology, Inc.; cat. no. 9272), p-Akt (S473; Cell Signaling Technology, Inc.; cat. no. 4060), PARP (Cell Signaling Technology, Inc.; cat. no. 9542), cleaved PARP (Cell Signaling Technology, Inc.; cat. no. 5625), caspase-3 (Cell Signaling Technology, Inc.; cat. no. 7148), -7 (Cell Signaling Technology, Inc.; cat. no. 12827) and -8 (Cell Signaling Technology, Inc.; cat. no. 4790), cleaved caspase-3 (Cell Signaling Technology, Inc.; cat. no. 9664), -7 (Cell Signaling Technology, Inc.; cat. no. 8438) and -8 (Cell Signaling Technology, Inc.; cat. no. 9496), Bax (Cell Signaling Technology, Inc.; cat. no. 2772) and vimentin (Cell Signaling Technology, Inc.; cat. no. 5741); anti-intracellular domain (ICD) HER2 clone 4B5 (Ventana Medical Systems; cat. no. 790-4493) and GAPDH (Invitrogen; Thermo Fisher Scientific, Inc.; cat. no. MA5-15738).

    Techniques: Immunofluorescence, Staining, Immunohistochemical staining, Fluorescence, Control

    A TTNT with T-DXd by HER2 status of the disease; B TTNT with T-DXd by clinical subtype of the disease; C TTNT with T-DXd depending on changes in HER2 status (low vs. 0) between the primary tumor and last metastatic biopsy; D TTNT with T-DXd depending on changes in HER2 status (low vs. 0) between the first and most recent metastatic biopsy; E OS with T-DXd by HER2 status of the disease; F OS with T-DXd by clinical subtype of the disease. T-DXd, trastuzumab deruxtecan; TTNT, time to next treatment; HER2, human epidermal growth factor receptor 2; OS, overall survival; HR, hormone receptor; TNBC, triple-negative breast cancer.

    Journal: NPJ Precision Oncology

    Article Title: Quantitative HER2 tissue and plasma profiling predicts the activity of trastuzumab deruxtecan for breast cancer

    doi: 10.1038/s41698-026-01365-6

    Figure Lengend Snippet: A TTNT with T-DXd by HER2 status of the disease; B TTNT with T-DXd by clinical subtype of the disease; C TTNT with T-DXd depending on changes in HER2 status (low vs. 0) between the primary tumor and last metastatic biopsy; D TTNT with T-DXd depending on changes in HER2 status (low vs. 0) between the first and most recent metastatic biopsy; E OS with T-DXd by HER2 status of the disease; F OS with T-DXd by clinical subtype of the disease. T-DXd, trastuzumab deruxtecan; TTNT, time to next treatment; HER2, human epidermal growth factor receptor 2; OS, overall survival; HR, hormone receptor; TNBC, triple-negative breast cancer.

    Article Snippet: The Leica BOND Rx autostainer protocol is as follows: deparaffinization with BOND dewax solution (AR9222), antigen retrieval with BOND HIER epitope retrieval solution 2 (AR9640) at 97 ° C for 20 min, blocking with ReadyProbes Endogenous HRP & AP blocking solution ( R37629 , Invitrogen) for 10 min and with BSA for 30 min, 1 h incubation with primary rabbit monoclonal HER2 antibody (clone 29D8, #2165, IgG, Cell Signaling) at optimal concentration of 1ug/ml mixed together with 1:100 concentration of pan-CK (Clones AE1/AE3, REF#M3515, Dako), amplification with Rabbit Envision+ System–HRP labeled polymer anti-Rabbit (K400311-2, Dako) mixed together with 1:100 dilution of a green-fluorescent Alexa Fluor 546 Goat-anti-Mouse IgG (H + L) Cross-Adsorbed Secondary Antibody (REF#A11003) for 1 h, staining with 1:50 dilution of a red-fluorescent Tyramide Signal Amplification (TSA) Cyanin 5 (SAT705A001EA, Akoya Biosciences) for 10 min and nuclear staining with 1:500 dilution of a blue-fluorescent 4′,6-diamidino-2-phenylindole (DAPI) for 10 min.

    Techniques:

    Samples are ordered by HER2 IHC subtype and TTNT from the start of T-DXd treatment. Clinical parameters displayed include HER2 IHC subtype (HER2-zero, HER2-low, HER2-positive), latest HER2 IHC prior to T-DXd, number of prior lines of chemotherapy, and receipt of sacituzumab govitecan before T-DXd. An asterisk on the TTNT bar indicates treatment is still ongoing. Bar plots show scores for each assay, and these values are also represented with color-coded squares, along with their corresponding quartiles for the HS-HER2 and RPPA HER2 assays. For HER2DX ERBB2, score groups are displayed based on the recommended cutoffs: Low (1–32), Med (33–50), and High (51–99) . Some cases are depicted as HER2-positive given prior HER2-positivity, despite the closest biopsy to T-DXd administration being formally HER2-negative (as suggested by the IHC score). HS-HER2, High Sensitivity-human epidermal growth factor receptor 2; RPPA, Reverse Phase Protein Array; T-DXd, trastuzumab deruxtecan; TTNT, time to next treatment. *did not experience TTNT event.

    Journal: NPJ Precision Oncology

    Article Title: Quantitative HER2 tissue and plasma profiling predicts the activity of trastuzumab deruxtecan for breast cancer

    doi: 10.1038/s41698-026-01365-6

    Figure Lengend Snippet: Samples are ordered by HER2 IHC subtype and TTNT from the start of T-DXd treatment. Clinical parameters displayed include HER2 IHC subtype (HER2-zero, HER2-low, HER2-positive), latest HER2 IHC prior to T-DXd, number of prior lines of chemotherapy, and receipt of sacituzumab govitecan before T-DXd. An asterisk on the TTNT bar indicates treatment is still ongoing. Bar plots show scores for each assay, and these values are also represented with color-coded squares, along with their corresponding quartiles for the HS-HER2 and RPPA HER2 assays. For HER2DX ERBB2, score groups are displayed based on the recommended cutoffs: Low (1–32), Med (33–50), and High (51–99) . Some cases are depicted as HER2-positive given prior HER2-positivity, despite the closest biopsy to T-DXd administration being formally HER2-negative (as suggested by the IHC score). HS-HER2, High Sensitivity-human epidermal growth factor receptor 2; RPPA, Reverse Phase Protein Array; T-DXd, trastuzumab deruxtecan; TTNT, time to next treatment. *did not experience TTNT event.

    Article Snippet: The Leica BOND Rx autostainer protocol is as follows: deparaffinization with BOND dewax solution (AR9222), antigen retrieval with BOND HIER epitope retrieval solution 2 (AR9640) at 97 ° C for 20 min, blocking with ReadyProbes Endogenous HRP & AP blocking solution ( R37629 , Invitrogen) for 10 min and with BSA for 30 min, 1 h incubation with primary rabbit monoclonal HER2 antibody (clone 29D8, #2165, IgG, Cell Signaling) at optimal concentration of 1ug/ml mixed together with 1:100 concentration of pan-CK (Clones AE1/AE3, REF#M3515, Dako), amplification with Rabbit Envision+ System–HRP labeled polymer anti-Rabbit (K400311-2, Dako) mixed together with 1:100 dilution of a green-fluorescent Alexa Fluor 546 Goat-anti-Mouse IgG (H + L) Cross-Adsorbed Secondary Antibody (REF#A11003) for 1 h, staining with 1:50 dilution of a red-fluorescent Tyramide Signal Amplification (TSA) Cyanin 5 (SAT705A001EA, Akoya Biosciences) for 10 min and nuclear staining with 1:500 dilution of a blue-fluorescent 4′,6-diamidino-2-phenylindole (DAPI) for 10 min.

    Techniques: Protein Array

    A Workflow for the quantification of HS-HER2; B TTNT with T-DXd according to HS-HER2 quartiles; C OS with T-DXd according to HS-HER2 quartiles; D TTNT with T-DXd according to HS-HER2 median (HER2-positive disease only); E TTNT with T-DXd in the HS-HER2 cohort according to the traditional HER2 IHC classification of HER2-positive, HER2-low-HER2-0 in the HS-HER2 population. T-DXd trastuzumab deruxtecan, TTNT time to next treatment, HS-HER2 High Sensitivity-HER2, IHC immunohistochemistry, ROI region of interest, OS overall survival.

    Journal: NPJ Precision Oncology

    Article Title: Quantitative HER2 tissue and plasma profiling predicts the activity of trastuzumab deruxtecan for breast cancer

    doi: 10.1038/s41698-026-01365-6

    Figure Lengend Snippet: A Workflow for the quantification of HS-HER2; B TTNT with T-DXd according to HS-HER2 quartiles; C OS with T-DXd according to HS-HER2 quartiles; D TTNT with T-DXd according to HS-HER2 median (HER2-positive disease only); E TTNT with T-DXd in the HS-HER2 cohort according to the traditional HER2 IHC classification of HER2-positive, HER2-low-HER2-0 in the HS-HER2 population. T-DXd trastuzumab deruxtecan, TTNT time to next treatment, HS-HER2 High Sensitivity-HER2, IHC immunohistochemistry, ROI region of interest, OS overall survival.

    Article Snippet: The Leica BOND Rx autostainer protocol is as follows: deparaffinization with BOND dewax solution (AR9222), antigen retrieval with BOND HIER epitope retrieval solution 2 (AR9640) at 97 ° C for 20 min, blocking with ReadyProbes Endogenous HRP & AP blocking solution ( R37629 , Invitrogen) for 10 min and with BSA for 30 min, 1 h incubation with primary rabbit monoclonal HER2 antibody (clone 29D8, #2165, IgG, Cell Signaling) at optimal concentration of 1ug/ml mixed together with 1:100 concentration of pan-CK (Clones AE1/AE3, REF#M3515, Dako), amplification with Rabbit Envision+ System–HRP labeled polymer anti-Rabbit (K400311-2, Dako) mixed together with 1:100 dilution of a green-fluorescent Alexa Fluor 546 Goat-anti-Mouse IgG (H + L) Cross-Adsorbed Secondary Antibody (REF#A11003) for 1 h, staining with 1:50 dilution of a red-fluorescent Tyramide Signal Amplification (TSA) Cyanin 5 (SAT705A001EA, Akoya Biosciences) for 10 min and nuclear staining with 1:500 dilution of a blue-fluorescent 4′,6-diamidino-2-phenylindole (DAPI) for 10 min.

    Techniques: Immunohistochemistry

    A Workflow for the CLIA-based RPPA analysis; B TTNT with T-DXd according to RPPA quantified total HER2 protein quartiles; C . OS with T-DXd according to RPPA HER2 protein expression quartiles; D TTNT with T-DXd according to RPPA measured HER2 activation (phosphoHER2 Y1248) quartiles; E OS with T-DXd according to RPPA measured HER2 activation (phosphoHER2 Y1248) quartiles; T-DXd trastuzumab deruxtecan, CLIA-RPPA Clinical Laboratory Improvement Amendments- Reverse Phase Protein Array, TTNT time to next treatment, OS overall survival.

    Journal: NPJ Precision Oncology

    Article Title: Quantitative HER2 tissue and plasma profiling predicts the activity of trastuzumab deruxtecan for breast cancer

    doi: 10.1038/s41698-026-01365-6

    Figure Lengend Snippet: A Workflow for the CLIA-based RPPA analysis; B TTNT with T-DXd according to RPPA quantified total HER2 protein quartiles; C . OS with T-DXd according to RPPA HER2 protein expression quartiles; D TTNT with T-DXd according to RPPA measured HER2 activation (phosphoHER2 Y1248) quartiles; E OS with T-DXd according to RPPA measured HER2 activation (phosphoHER2 Y1248) quartiles; T-DXd trastuzumab deruxtecan, CLIA-RPPA Clinical Laboratory Improvement Amendments- Reverse Phase Protein Array, TTNT time to next treatment, OS overall survival.

    Article Snippet: The Leica BOND Rx autostainer protocol is as follows: deparaffinization with BOND dewax solution (AR9222), antigen retrieval with BOND HIER epitope retrieval solution 2 (AR9640) at 97 ° C for 20 min, blocking with ReadyProbes Endogenous HRP & AP blocking solution ( R37629 , Invitrogen) for 10 min and with BSA for 30 min, 1 h incubation with primary rabbit monoclonal HER2 antibody (clone 29D8, #2165, IgG, Cell Signaling) at optimal concentration of 1ug/ml mixed together with 1:100 concentration of pan-CK (Clones AE1/AE3, REF#M3515, Dako), amplification with Rabbit Envision+ System–HRP labeled polymer anti-Rabbit (K400311-2, Dako) mixed together with 1:100 dilution of a green-fluorescent Alexa Fluor 546 Goat-anti-Mouse IgG (H + L) Cross-Adsorbed Secondary Antibody (REF#A11003) for 1 h, staining with 1:50 dilution of a red-fluorescent Tyramide Signal Amplification (TSA) Cyanin 5 (SAT705A001EA, Akoya Biosciences) for 10 min and nuclear staining with 1:500 dilution of a blue-fluorescent 4′,6-diamidino-2-phenylindole (DAPI) for 10 min.

    Techniques: Expressing, Activation Assay, Protein Array

    A TTNT with T-DXd according to RPPA measured TOPO1 protein expression median (HER2-negative only); B OS with T-DXd according to RPPA measured TOPO1 protein expression median (HER2-negative only); C TTNT with T-DXd according to RPPA Trop2 quartiles; D TTNT with T-DXd according to RPPA measured EGFR protein expression quartiles; E TTNT with T-DXd according to RPPA measured phosphoHER3 protein expression quartiles; F TTNT with T-DXd according to the traditional HER2 IHC classification of HER2-positive, HER2-low-HER2-0 in the RPPA population. T-DXd trastuzumab deruxtecan, TTNT time to next treatment, RPPA Reverse Phase Protein Array, OS overall survival.

    Journal: NPJ Precision Oncology

    Article Title: Quantitative HER2 tissue and plasma profiling predicts the activity of trastuzumab deruxtecan for breast cancer

    doi: 10.1038/s41698-026-01365-6

    Figure Lengend Snippet: A TTNT with T-DXd according to RPPA measured TOPO1 protein expression median (HER2-negative only); B OS with T-DXd according to RPPA measured TOPO1 protein expression median (HER2-negative only); C TTNT with T-DXd according to RPPA Trop2 quartiles; D TTNT with T-DXd according to RPPA measured EGFR protein expression quartiles; E TTNT with T-DXd according to RPPA measured phosphoHER3 protein expression quartiles; F TTNT with T-DXd according to the traditional HER2 IHC classification of HER2-positive, HER2-low-HER2-0 in the RPPA population. T-DXd trastuzumab deruxtecan, TTNT time to next treatment, RPPA Reverse Phase Protein Array, OS overall survival.

    Article Snippet: The Leica BOND Rx autostainer protocol is as follows: deparaffinization with BOND dewax solution (AR9222), antigen retrieval with BOND HIER epitope retrieval solution 2 (AR9640) at 97 ° C for 20 min, blocking with ReadyProbes Endogenous HRP & AP blocking solution ( R37629 , Invitrogen) for 10 min and with BSA for 30 min, 1 h incubation with primary rabbit monoclonal HER2 antibody (clone 29D8, #2165, IgG, Cell Signaling) at optimal concentration of 1ug/ml mixed together with 1:100 concentration of pan-CK (Clones AE1/AE3, REF#M3515, Dako), amplification with Rabbit Envision+ System–HRP labeled polymer anti-Rabbit (K400311-2, Dako) mixed together with 1:100 dilution of a green-fluorescent Alexa Fluor 546 Goat-anti-Mouse IgG (H + L) Cross-Adsorbed Secondary Antibody (REF#A11003) for 1 h, staining with 1:50 dilution of a red-fluorescent Tyramide Signal Amplification (TSA) Cyanin 5 (SAT705A001EA, Akoya Biosciences) for 10 min and nuclear staining with 1:500 dilution of a blue-fluorescent 4′,6-diamidino-2-phenylindole (DAPI) for 10 min.

    Techniques: Expressing, Protein Array

    A Description of HER2DX gene expression modules; association between the HER2 amplicon module with TTNT ( B ) and OS in all patients ( C ); association between the HER2 amplicon module with TTNT ( D ) and OS in HER2-positive MBC ( E ); F association between TTNT and the luminal module in patients with HER2-negative disease; G TTNT with T-DXd according to the traditional HER2 IHC classification of HER2-positive, HER2-low-HER2-0 in the HER2DX population. TTNT time to next treatment, OS overall survival, MBC metastatic breast cancer, T-DXd trastuzumab deruxtecan, IHC immunohistochemistry.

    Journal: NPJ Precision Oncology

    Article Title: Quantitative HER2 tissue and plasma profiling predicts the activity of trastuzumab deruxtecan for breast cancer

    doi: 10.1038/s41698-026-01365-6

    Figure Lengend Snippet: A Description of HER2DX gene expression modules; association between the HER2 amplicon module with TTNT ( B ) and OS in all patients ( C ); association between the HER2 amplicon module with TTNT ( D ) and OS in HER2-positive MBC ( E ); F association between TTNT and the luminal module in patients with HER2-negative disease; G TTNT with T-DXd according to the traditional HER2 IHC classification of HER2-positive, HER2-low-HER2-0 in the HER2DX population. TTNT time to next treatment, OS overall survival, MBC metastatic breast cancer, T-DXd trastuzumab deruxtecan, IHC immunohistochemistry.

    Article Snippet: The Leica BOND Rx autostainer protocol is as follows: deparaffinization with BOND dewax solution (AR9222), antigen retrieval with BOND HIER epitope retrieval solution 2 (AR9640) at 97 ° C for 20 min, blocking with ReadyProbes Endogenous HRP & AP blocking solution ( R37629 , Invitrogen) for 10 min and with BSA for 30 min, 1 h incubation with primary rabbit monoclonal HER2 antibody (clone 29D8, #2165, IgG, Cell Signaling) at optimal concentration of 1ug/ml mixed together with 1:100 concentration of pan-CK (Clones AE1/AE3, REF#M3515, Dako), amplification with Rabbit Envision+ System–HRP labeled polymer anti-Rabbit (K400311-2, Dako) mixed together with 1:100 dilution of a green-fluorescent Alexa Fluor 546 Goat-anti-Mouse IgG (H + L) Cross-Adsorbed Secondary Antibody (REF#A11003) for 1 h, staining with 1:50 dilution of a red-fluorescent Tyramide Signal Amplification (TSA) Cyanin 5 (SAT705A001EA, Akoya Biosciences) for 10 min and nuclear staining with 1:500 dilution of a blue-fluorescent 4′,6-diamidino-2-phenylindole (DAPI) for 10 min.

    Techniques: Gene Expression, Amplification, Immunohistochemistry

    A Description of DNADX workflow; B Outcomes with T-DXd for metastatic breast cancer according to DNADX-detected tumor fraction; C Outcomes with T-DXd for metastatic breast cancer according to DNADX subtype; D Outcomes with T-DXd for metastatic breast cancer according to the DNADX HER2 signature; E Outcomes with T-DXd for metastatic breast cancer according to HER2 IHC status in the cohort of patients with detectable tumor fraction. T-DXd trastuzumab deruxtecan, TTNT, time to next treatment, OS overall survival.

    Journal: NPJ Precision Oncology

    Article Title: Quantitative HER2 tissue and plasma profiling predicts the activity of trastuzumab deruxtecan for breast cancer

    doi: 10.1038/s41698-026-01365-6

    Figure Lengend Snippet: A Description of DNADX workflow; B Outcomes with T-DXd for metastatic breast cancer according to DNADX-detected tumor fraction; C Outcomes with T-DXd for metastatic breast cancer according to DNADX subtype; D Outcomes with T-DXd for metastatic breast cancer according to the DNADX HER2 signature; E Outcomes with T-DXd for metastatic breast cancer according to HER2 IHC status in the cohort of patients with detectable tumor fraction. T-DXd trastuzumab deruxtecan, TTNT, time to next treatment, OS overall survival.

    Article Snippet: The Leica BOND Rx autostainer protocol is as follows: deparaffinization with BOND dewax solution (AR9222), antigen retrieval with BOND HIER epitope retrieval solution 2 (AR9640) at 97 ° C for 20 min, blocking with ReadyProbes Endogenous HRP & AP blocking solution ( R37629 , Invitrogen) for 10 min and with BSA for 30 min, 1 h incubation with primary rabbit monoclonal HER2 antibody (clone 29D8, #2165, IgG, Cell Signaling) at optimal concentration of 1ug/ml mixed together with 1:100 concentration of pan-CK (Clones AE1/AE3, REF#M3515, Dako), amplification with Rabbit Envision+ System–HRP labeled polymer anti-Rabbit (K400311-2, Dako) mixed together with 1:100 dilution of a green-fluorescent Alexa Fluor 546 Goat-anti-Mouse IgG (H + L) Cross-Adsorbed Secondary Antibody (REF#A11003) for 1 h, staining with 1:50 dilution of a red-fluorescent Tyramide Signal Amplification (TSA) Cyanin 5 (SAT705A001EA, Akoya Biosciences) for 10 min and nuclear staining with 1:500 dilution of a blue-fluorescent 4′,6-diamidino-2-phenylindole (DAPI) for 10 min.

    Techniques: