ihc cst 2165s ab 10692490 phospho her3 erbb3 tyr1289  (Cell Signaling Technology Inc)

Journal: Scientific Reports

Article Title: Pyrotinib promotes the antitumor effect of T-DM1 by increasing drug endocytosis in HER2-positive breast cancer

doi: 10.1038/s41598-025-03678-1

Figure Lengend Snippet: Pyrotinib downregulates HER2 protein levels and suppresses phosphorylation of HER2, PI3K/AKT, and RAS/MAPK signaling pathways in SK-BR-3 and JIMT-1 cells. ( a – d ) Expression levels of HER2 and its downstream proteins in the PI3K/AKT and RAS/MAPK signaling pathways were analyzed by western blotting. ( a ) Both cell types were treated with lapatinib (0.001, 0.01, 0.1, 1 µM) for 24 h. ( b ) Both cell types were treated with lapatinib (1 µM) at different time points. ( c ) Both cell types were treated with pyrotinib (0.001, 0.01, 0.1, 1 µM) for 24 h. ( d ) Both cell types were treated with pyrotinib (0.1 µM) at different time points. Results are representative of 3 independent replicates.

Article Snippet: After blocking with 5% nonfat milk or 3% bovine serum albumin (BSA), the membranes were incubated at 4 °C overnight with the primary antibodies HER2 Rabbit mAb, Phospho-HER2 (Tyr1221/1222) Rabbit mAb, Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) Rabbit mAb, and Phospho-Akt (Ser473) Rabbit mAb, which were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Phospho-proteomics, Protein-Protein interactions, Expressing, Western Blot

Journal: Scientific Reports

Article Title: Pyrotinib promotes the antitumor effect of T-DM1 by increasing drug endocytosis in HER2-positive breast cancer

doi: 10.1038/s41598-025-03678-1

Figure Lengend Snippet: Pyrotinib promotes HER2 degradation via the ubiquitin–proteasome pathway. ( a ) HER2 mRNA expression in SK-BR-3 and JIMT-1 cells treated with pyrotinib (0.5µM) or lapatinib (1µM) as assessed using RT-qPCR. ( b – d ) SK-BR-3 and JIMT-1 cells were treated with the lysosomal inhibitor Baf-A1 (20nM) or proteasome inhibitors Velcade (0.5 µM) or MG-132 (10µM) for 0.5 h, with DMSO as the control, followed by the addition of pyrotinib (0.5µM) for 0, 2, and 4 h. ( e – f ) Cells were subjected to MG132 (10 µM) treatment for 0.5 h or DMSO as the control, followed by the addition of pyrotinib (0.5 µM) for 0, 2, and 4 h. HER2 was immunoprecipitated from the lysates, and the samples were analyzed by immunoblotting with an anti-ubiquitin, anti-HER2, and anti-HSP70 antibodies. GAPDH served as the loading control. Results are representative of 3 independent replicates.

Article Snippet: After blocking with 5% nonfat milk or 3% bovine serum albumin (BSA), the membranes were incubated at 4 °C overnight with the primary antibodies HER2 Rabbit mAb, Phospho-HER2 (Tyr1221/1222) Rabbit mAb, Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) Rabbit mAb, and Phospho-Akt (Ser473) Rabbit mAb, which were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Ubiquitin Proteomics, Expressing, Quantitative RT-PCR, Control, Immunoprecipitation, Western Blot

Journal: Scientific Reports

Article Title: Pyrotinib promotes the antitumor effect of T-DM1 by increasing drug endocytosis in HER2-positive breast cancer

doi: 10.1038/s41598-025-03678-1

Figure Lengend Snippet: Pyrotinib promotes HER2 internalization and T-DM1 endocytosis. ( a ) Cells were treated with pyrotinib (0.5µM) or lapatinib (1 µM) for 0, 2, and 4 h and processed for immunofluorescence experiments using anti-HER2 antibody (green). Nuclei were stained with DAPI (blue) (×1000), Scale bar = 10 μm. ( b ) After labeling T-DM1 with pHrodo Deep Red (pHro-do-T-DM1), the cells were exposed to pHrodo-T-DM1 (1 µg/mL) alone or in combination with pyrotinib (0.1µM) for 0, 2, and 4 h. pHrodo-T-DM1 emits red fluorescent signals within the cellular interior. Nuclei were stained with DAPI (blue) (×600). The quantification of T-DM1 fluorescence intensity is now shown in Supplementary Figure . Scale bar = 10 μm. Results are representative of 3 independent replicates.

Article Snippet: After blocking with 5% nonfat milk or 3% bovine serum albumin (BSA), the membranes were incubated at 4 °C overnight with the primary antibodies HER2 Rabbit mAb, Phospho-HER2 (Tyr1221/1222) Rabbit mAb, Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) Rabbit mAb, and Phospho-Akt (Ser473) Rabbit mAb, which were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Immunofluorescence, Staining, Labeling, Fluorescence

Journal: Scientific Reports

Article Title: Pyrotinib promotes the antitumor effect of T-DM1 by increasing drug endocytosis in HER2-positive breast cancer

doi: 10.1038/s41598-025-03678-1

Figure Lengend Snippet: Pyrotinib enhances the antitumor effect of T-DM1 in vivo. ( a ) Images of JIMT-1 xenografts harvested after 21 days of treatment with T-DM1 (10 mg/kg) with or without pyrotinib (2 mg/kg) ( n = 6). ( b ) Changes in tumor weight in the examined mice. ( c ) Changes in tumor volume in the examined mice. ( d ) Body weight changes in mice after treatments. ( e ) Representative images displaying HE and IHC staining of xenograft tumor tissues (×400), scale bars = 50 μm. The histogram shows the average absorbance of HER2. ( f ) The protein expression levels of HER2 and its downstream signaling pathways in tumor tissues of each group were analyzed by western blot. GAPDH served as the loading control. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns = not significant. Results are representative of 3 independent replicates.

Article Snippet: After blocking with 5% nonfat milk or 3% bovine serum albumin (BSA), the membranes were incubated at 4 °C overnight with the primary antibodies HER2 Rabbit mAb, Phospho-HER2 (Tyr1221/1222) Rabbit mAb, Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) Rabbit mAb, and Phospho-Akt (Ser473) Rabbit mAb, which were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: In Vivo, Immunohistochemistry, Expressing, Protein-Protein interactions, Western Blot, Control