anti phospho her2 erbb2  (Cell Signaling Technology Inc)

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

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

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

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

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

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

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

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

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

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

Journal: Journal of Extracellular Biology

Article Title: A One‐Step Workflow for Size‐Based Separation of Extracellular Vesicles With Integrated Surface Marker Detection

doi: 10.1002/jex2.70109

Figure Lengend Snippet: Representation and validation of the AF4‐MALS‐FLD method . (A) Overview of the workflow used for identification of EV surface proteins. PE‐conjugated antibodies were incubated with the sample (e.g. pre‐purified EVs, cell culture supernatant, urine, or plasma) and loaded into the AF4 channel. (B) The light scatter elution profile (in relative scale) (black, full line), UV elution profile (black, dotted line) and the size determination ( R rms in nm) (red) obtained by the multi‐angle light scattering (MALS) detector is plotted against time for labelling of SK‐BR‐3‐derived EVs with PE‐conjugated anti‐CD81 antibody. (C) The fluorescent light detector (FLD) signal (in relative scale) for SK‐BR‐3‐derived EVs labelled with PE‐conjugated anti‐CD9, anti‐CD63 and anti‐CD81 is plotted against time. (D) Transmission electron microscopy (TEM) images of different fractions of the AF4‐MALS‐FLD elution profile are shown (scale bar = 200 nm).

Article Snippet: The following primary and secondary antibodies were used for western blot analysis: mouse monoclonal anti‐Alix (1:1000) (cat no. 2171S, Cell Signaling Technology), rabbit monoclonal anti‐CD9 (1:1000) (cat no. 13403S, Cell Signaling Technology), rabbit monoclonal anti‐Syntenin‐1 (1:1000) (cat no. ab133267, Abcam), mouse monoclonal anti‐TSG101 (1:1000) (cat no. sc‐7964, Santa Cruz Biotechnology), rabbit monoclonal anti‐PSMA (1:1000) (cat no. 12702S), mouse monoclonal anti‐EpCAM (1:1000) (cat no. 2929S, Cell Signaling Technology), rabbit monoclonal anti‐HER2 (1:1000) (cat no. 2165S, Cell Signaling Technology), mouse monoclonal anti‐GAPDH (1:2500) (cat no. G8795, Merck Life Science), sheep anti‐mouse horseradish peroxidase‐linked (1:3000) (cat no. NA931V, GE Healthcare Life Sciences) and donkey anti‐rabbit horseradish peroxidase‐linked antibody (1:8000) (cat no. NA934V, GE Healthcare Life Sciences).

Techniques: Biomarker Discovery, Incubation, Purification, Cell Culture, Clinical Proteomics, Multi-Angle Light Scattering, Derivative Assay, Transmission Assay, Electron Microscopy

Journal: Journal of Extracellular Biology

Article Title: A One‐Step Workflow for Size‐Based Separation of Extracellular Vesicles With Integrated Surface Marker Detection

doi: 10.1002/jex2.70109

Figure Lengend Snippet: AF4‐MALS‐FLD analysis of EV surface proteins with biomarker potential in prostate and breast cancer . MCF‐7‐, MDA‐MB‐231‐ and SK‐BR‐3‐derived EVs were labelled with PE‐conjugated anti‐EpCAM antibodies and analysed by AF4‐MALS‐FLD. (A) The elution profile (in relative scale) of the multi‐angle light scatter (MALS) detector and the size ( R rms in nm) were plotted against time. The fluorescent light detector (FLD) signal for MCF‐7‐, MDA‐MB‐231‐ and SK‐BR‐3‐derived EVs labelled with (B) PE‐conjugated anti‐EpCAM and (C) PE‐conjugated anti‐HER2 antibodies were plotted. (D) From FLD elution profiles, the area under the curve for the EV peak (24–80 min) was determined. Unstained EV samples were used as a negative control. (E) Different concentrations (6 × 10 9 , 8 × 10 9 , 1 × 10 10 and 2 × 10 10 particles as measured by NTA) including a negative control of LNCaP‐derived EVs (high PSMA expression) were labelled with anti‐PSMA antibodies and analysed by the AF4‐MALS‐FLD protocol. (F) The area under the curve for the EV peak was determined for LNCaP‐derived EVs. Different concentrations (2 × 10 10 , 4 × 10 10 and 6 × 10 10 particles as measured by NTA) including a negative control of (G) MCF‐7‐derived EVs (high EpCAM expression) or (I) SK‐BR‐3‐derived EVs (high HER2 expression) were labelled with PE‐conjugated anti‐EpCAM or anti‐HER2 antibodies respectively and analysed by the AF4‐MALS‐FLD protocol. The area under the curve for the EV peak (24–80 min) was determined for (H) MCF‐7‐ and (J) SK‐BR‐3‐derived EVs.

Article Snippet: The following primary and secondary antibodies were used for western blot analysis: mouse monoclonal anti‐Alix (1:1000) (cat no. 2171S, Cell Signaling Technology), rabbit monoclonal anti‐CD9 (1:1000) (cat no. 13403S, Cell Signaling Technology), rabbit monoclonal anti‐Syntenin‐1 (1:1000) (cat no. ab133267, Abcam), mouse monoclonal anti‐TSG101 (1:1000) (cat no. sc‐7964, Santa Cruz Biotechnology), rabbit monoclonal anti‐PSMA (1:1000) (cat no. 12702S), mouse monoclonal anti‐EpCAM (1:1000) (cat no. 2929S, Cell Signaling Technology), rabbit monoclonal anti‐HER2 (1:1000) (cat no. 2165S, Cell Signaling Technology), mouse monoclonal anti‐GAPDH (1:2500) (cat no. G8795, Merck Life Science), sheep anti‐mouse horseradish peroxidase‐linked (1:3000) (cat no. NA931V, GE Healthcare Life Sciences) and donkey anti‐rabbit horseradish peroxidase‐linked antibody (1:8000) (cat no. NA934V, GE Healthcare Life Sciences).

Techniques: Biomarker Discovery, Derivative Assay, Multi-Angle Light Scattering, Negative Control, Expressing

Journal: Journal of Extracellular Biology

Article Title: A One‐Step Workflow for Size‐Based Separation of Extracellular Vesicles With Integrated Surface Marker Detection

doi: 10.1002/jex2.70109

Figure Lengend Snippet: Detection of EVs in complex matrices . (A) Different volumes of cell culture supernatant (0, 20, 40 and 60 µL) collected from the MCF‐7 cells were labelled with PE‐conjugated anti‐EpCAM antibodies and analysed by AF4‐MALS‐FLD. The area under the curve for the EV peak in complex matrices (40–80 min) was determined. (B) Different amounts of LNCaP‐derived EVs were spiked in 100 µL of concentrated urine, diluted 1:1 in PBS to reduce viscosity, labelled with PE‐conjugated anti‐PSMA antibodies, and analysed by AF4‐MALS‐FLD. The area under the curve for the EV peak was determined. Different amounts of (C) MCF‐7‐ or (D) SK‐BR‐3‐derived EVs were spiked in 100 µL of blood plasma, diluted 1:1 in PBS to reduce viscosity, and labelled with PE‐conjugated anti‐EpCAM or anti‐HER2 antibodies, respectively. Labelled EVs were analysed by AF4‐MALS‐FLD and the area under the curve for the EV peak was determined. Different amounts of SK‐BR‐3 EVs were also spiked in blood plasma and labelled with isotype control antibodies. (E) Different concentrations of soluble EpCAM (1, 5 and 10 ng/mL) and soluble HER2 (50, 100 and 150 ng/mL) were spiked in blood plasma, labelled with PE‐conjugated anti‐EpCAM or anti‐HER2 antibodies respectively, and analysed by AF4‐MALS‐FLD.

Article Snippet: The following primary and secondary antibodies were used for western blot analysis: mouse monoclonal anti‐Alix (1:1000) (cat no. 2171S, Cell Signaling Technology), rabbit monoclonal anti‐CD9 (1:1000) (cat no. 13403S, Cell Signaling Technology), rabbit monoclonal anti‐Syntenin‐1 (1:1000) (cat no. ab133267, Abcam), mouse monoclonal anti‐TSG101 (1:1000) (cat no. sc‐7964, Santa Cruz Biotechnology), rabbit monoclonal anti‐PSMA (1:1000) (cat no. 12702S), mouse monoclonal anti‐EpCAM (1:1000) (cat no. 2929S, Cell Signaling Technology), rabbit monoclonal anti‐HER2 (1:1000) (cat no. 2165S, Cell Signaling Technology), mouse monoclonal anti‐GAPDH (1:2500) (cat no. G8795, Merck Life Science), sheep anti‐mouse horseradish peroxidase‐linked (1:3000) (cat no. NA931V, GE Healthcare Life Sciences) and donkey anti‐rabbit horseradish peroxidase‐linked antibody (1:8000) (cat no. NA934V, GE Healthcare Life Sciences).

Techniques: Cell Culture, Derivative Assay, Viscosity, Clinical Proteomics, Control

Journal: Journal of Extracellular Biology

Article Title: A One‐Step Workflow for Size‐Based Separation of Extracellular Vesicles With Integrated Surface Marker Detection

doi: 10.1002/jex2.70109

Figure Lengend Snippet: Validation of the AF4‐MALS‐FLD workflow on patient samples . Urine samples of five prostate cancer patients were labelled for PSMA and analysed by the AF4‐MALS‐FLD workflow. Fractions 40–80 min were collected, concentrated and processed for mass spectrometry‐based proteomic analysis. (A) EV markers Syntenin‐1, Flotillin‐1, CD63, CD9, CD81, Flotillin‐2, Alix and TSG101 were analysed (missing sample indicated in grey). Z ‐score transformation of intensities were plotted. (B) Targeted mass spectrometry analysed the presence of PSMA (FOLH1) in patient samples. The z ‐score transformation of intensities was plotted with the AF4‐MALS‐FLD peak area. (C) Blood plasma samples of healthy controls ( n = 7) and HER2 amplified breast cancer patients ( n = 10) were labelled with PE‐conjugated anti‐HER2 antibodies. (D) Blood plasma samples of healthy controls ( n = 6) and breast cancer patients ( n = 8) were labelled with PE‐conjugated anti‐EpCAM antibodies. The area under the curve values were normalised for the mean value in the healthy control group.

Article Snippet: The following primary and secondary antibodies were used for western blot analysis: mouse monoclonal anti‐Alix (1:1000) (cat no. 2171S, Cell Signaling Technology), rabbit monoclonal anti‐CD9 (1:1000) (cat no. 13403S, Cell Signaling Technology), rabbit monoclonal anti‐Syntenin‐1 (1:1000) (cat no. ab133267, Abcam), mouse monoclonal anti‐TSG101 (1:1000) (cat no. sc‐7964, Santa Cruz Biotechnology), rabbit monoclonal anti‐PSMA (1:1000) (cat no. 12702S), mouse monoclonal anti‐EpCAM (1:1000) (cat no. 2929S, Cell Signaling Technology), rabbit monoclonal anti‐HER2 (1:1000) (cat no. 2165S, Cell Signaling Technology), mouse monoclonal anti‐GAPDH (1:2500) (cat no. G8795, Merck Life Science), sheep anti‐mouse horseradish peroxidase‐linked (1:3000) (cat no. NA931V, GE Healthcare Life Sciences) and donkey anti‐rabbit horseradish peroxidase‐linked antibody (1:8000) (cat no. NA934V, GE Healthcare Life Sciences).

Techniques: Biomarker Discovery, Mass Spectrometry, Transformation Assay, Clinical Proteomics, Amplification, Control