phospho her2 tyr1221 1222 rabbit mab  (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

Journal: Breast cancer research : BCR

Article Title: Erbin interacts with NHERF1 and Ezrin to stabilize a membrane ErbB2 signaling complex in HER2-positive breast cancer.

doi: 10.1186/s13058-025-02025-6

Figure Lengend Snippet: Fig. 6 Ezrin and NHERF1 are required for Erbin and HER2 interactions. A) Immunofluorescence for Erbin with HER2 (top row), NHERF1 (middle row), and Ezrin (bottom row) in NSC668394 treated SKBR3 cells. All scale bars represent 10µM. B) Immunofluorescence for Erbin with HER2 (top row) and Ezrin (bot tom row) in NHERF1 knockdown SKBR3 cells. All scale bars represent 10µM. C) PLA fluorescence showing protein-protein interactions between HER2/ Erbin, Erbin/NHERF1, or Erbin/Ezrin in control, NSC668394 treated-, and NHERF1 knock-down SKBR3 cells. All scale bars represent 10µM. (n = 3) D) Quan tification of PLA results was performed by measuring the fluorescent intensity of PLA signals at the cell surface. HER2/Erbin (control n = 14, NSC668394 n = 14, NHERF1KD n = 14), Erbin/NHERF1 (control n = 19, NSC668394 n = 19, NHERF1KD n = 19), Erbin/EZRIN (control n = 15, NSC668394 n = 15, NHERF1KD n = 15). Bar graphs represent the mean ± SEM. **** denotes p < 0.00005. (n = 3). E) A working model proposing how HER2 mediated disruption of apical/ basal polarity creates the opportunity for the formation of a multi-protein signaling complex that includes HER2, Erbin, PMCA2, NHERF1, and Ezrin. Cre ated with BioRender.com

Article Snippet: Primary antibodies were against: HER2 (sc33684), mouse β-actin (sc-69879) from Santa Cruz (Dallas, TX); EGFR (4267), phospho-EGFR (2234), phospho-HER2 (2247) from cell signaling (Danvers, MA).

Techniques: Immunofluorescence, Knockdown, Fluorescence, Protein-Protein interactions, Control, Disruption