anti human her2 rabbit polyclonal antibody  (Cell Signaling Technology Inc)

Journal: Antibodies

Article Title: Molecular Mechanism of HER2 Rapid Internalization and Redirected Trafficking Induced by Anti-HER2 Biparatopic Antibody

doi: 10.3390/antib9030049

Figure Lengend Snippet: Rapid internalization of anti-HER2-Bs compared with trastuzumab or 39S in BT-474 cells. Internalization time courses of trastuzumab-AF647 ( A – C ), 39S-AF647 ( D – F ), and anti-HER2-Bs-AF647 ( G – I ) in cells stained with cytoplasmic dye, CFSE) (green), are shown. Before the initiation of internalization, fluorescence antibodies are localized on the cell surface ( A , D , E ). At 30 min, both trastuzumab-AF647 (red) and 39S-AF647 (red) are slowly internalized into the cells ( B , E ), while a majority of anti-HER2-Bs-AF647 (red) are already internalized ( H ). At 2 h, the surface-bound anti-HER2-Bs-AF647 is completely internalized ( I ), while both trastuzumab-AF647 and 39S-AF647 remain on the cell surface ( C , F ). ( J ) Antibody internalization time course was quantified by an image analysis algorithm. Data are plotted as mean ± standard deviation from 6 wells. Scale bar is 10 μm.

Article Snippet: Rabbit anti-human HER2 polyclonal antibody (Cat# A048529-2) was purchased from Dako, Agilent Technologies (Santa Clara, CA, USA).

Techniques: Staining, Fluorescence, Standard Deviation

Journal: Antibodies

Article Title: Molecular Mechanism of HER2 Rapid Internalization and Redirected Trafficking Induced by Anti-HER2 Biparatopic Antibody

doi: 10.3390/antib9030049

Figure Lengend Snippet: Effect of dynamin and clathrin inhibitors on anti-HER2-Bs and trastuzumab internalization. Overlays of anti-HER2-Bs-AF647 ( A – F ) and trastuzumab-AF647 ( G – L ) with the cytoplasm dye (green) in BT-474 cells treated with either DMSO ( A , D , G , J ), dynamin inhibitor Dyngo 4a ( B , E , H , K ), or clathrin light chain inhibitor Pitstop 2 ( C , F , I , L ) are shown. At T = 0, anti-HER2-Bs-AF647 ( A , B , C ) or trastuzumab-AF647 ( G , H , I ) was bound on the surface of the treated cells. At 2 h, the internalization of both anti-HER2-Bs-AF647 (red) and trastuzumab-AF647 (red) was inhibited in the presence of Dyngo 4a (compare B , E to A , D for anti-HER2-Bs; compare H , K to G , J for trastuzumab) or Pitstop 2 (compare C , F to A , D for anti-HER2-Bs; compare I , L to G , J for trastuzumab). Scale bar is 10 μm.

Article Snippet: Rabbit anti-human HER2 polyclonal antibody (Cat# A048529-2) was purchased from Dako, Agilent Technologies (Santa Clara, CA, USA).

Techniques:

Journal: Antibodies

Article Title: Molecular Mechanism of HER2 Rapid Internalization and Redirected Trafficking Induced by Anti-HER2 Biparatopic Antibody

doi: 10.3390/antib9030049

Figure Lengend Snippet: The combination of antibodies did not accelerate internalization. However, the addition of a secondary antibody accelerated trastuzumab internalization. Combined treatment with trastuzumab-AF647 (red) and unlabeled 39S ( C ) did not accelerate internalization of trastuzumab compared to trastuzumab-AF647 alone at 1 h ( A ). The internalization rate of trastuzumab-AF647 in the presence of 39S ( C ) is similar to the single antibody treatment with trastuzumab-AF647 ( A ) or 39S-AF647 (red) ( B ). By contrast, and similar to the previous figures, anti-HER2-Bs-AF647 (red) internalized rapidly into the cells ( E ). ( D ) Addition of anti-human IgG accelerated the internalization of trastuzumab compared to the untreated cells (compare D to A ). However, the internalization rate of anti-HER2-Bs is still much more pronounced compared to the trastuzumab-AF647/anti-human IgG-treated cells (compare E to D ). Cytoplasm dye, CFSE, is shown in green. Scale bar is 10 μm.

Article Snippet: Rabbit anti-human HER2 polyclonal antibody (Cat# A048529-2) was purchased from Dako, Agilent Technologies (Santa Clara, CA, USA).

Techniques:

Journal: Antibodies

Article Title: Molecular Mechanism of HER2 Rapid Internalization and Redirected Trafficking Induced by Anti-HER2 Biparatopic Antibody

doi: 10.3390/antib9030049

Figure Lengend Snippet: Blocking one epitope on HER2 receptors slowed down the internalization of anti-HER2-Bs-AF647 significantly. Cytoplasm dye, CFSE, is shown in green. Similar to the previous figures shown, anti-HER2-Bs-AF647 (red) internalized rapidly ( C ) compared to either trastuzumab-AF647 (red) ( A ) or 39S-AF647 (red) ( B ) at 2 h. ( D ) By contrast, blocking of trastuzumab-binding epitopes significantly slowed down the internalization of anti-HER2-Bs-AF647 at 2 h (compare D to C ) and resembled the internalization of 39S alone (compare D to B ). ( E ) Similarly, blocking of the other epitopes (39S-binding epitopes) also slowed down the anti-HER2-Bs-AF647 internalization (compare E to C ). Note that at the start of internalization (T = 0), similar fluorescent signals (AF647) were observed for all tested conditions ( A – E ) (see ). Scale bar is 10 μm.

Article Snippet: Rabbit anti-human HER2 polyclonal antibody (Cat# A048529-2) was purchased from Dako, Agilent Technologies (Santa Clara, CA, USA).

Techniques: Blocking Assay, Binding Assay

Journal: Antibodies

Article Title: Molecular Mechanism of HER2 Rapid Internalization and Redirected Trafficking Induced by Anti-HER2 Biparatopic Antibody

doi: 10.3390/antib9030049

Figure Lengend Snippet: Anti-HER2-Bs, but not trastuzumab, 39S, or both in combination, induced efficient HER2 degradation. ( A ) The level of total HER2 was not changed in the cells treated with trastuzumab, 39S, or both in combination. However, treatment with anti-HER2-Bs resulted in a decrease in total HER2 expression by 6 h. ( B ) Anti-HER2-Bs induces HER2 receptor ubiquitination, and this coincides with HER2 degradation. Treatment with anti-HER2-Bs resulted in an increase in total HER2 ubiquitination at 1–2 h and K63-specific ubiquitination at 1 h. ( C ) Quantification of HER2 total ubiquitination level normalized to total HER2 receptor level. The normalized level of HER2 total ubiquitination increased from 30 min post treatment and was sustained during 2–6 h post treatment.

Article Snippet: Rabbit anti-human HER2 polyclonal antibody (Cat# A048529-2) was purchased from Dako, Agilent Technologies (Santa Clara, CA, USA).

Techniques: Expressing

Journal: Antibodies

Article Title: Molecular Mechanism of HER2 Rapid Internalization and Redirected Trafficking Induced by Anti-HER2 Biparatopic Antibody

doi: 10.3390/antib9030049

Figure Lengend Snippet: Anti-HER2-Bs and trastuzumab mediated distinct intracellular trafficking of internalized HER2 receptors. Internalization time courses of anti-HER2-Bs-AF647 ( A – C ) and trastuzumab-AF647 ( D – F ) are shown. ( A , D ) Before the initiation of internalization, both anti-HER2-Bs-AF647 and trastuzumab-AF647 (red) were colocalized with HER2 receptors on the cell surface (green) and appeared yellow. ( B , E ) At 30 min, the antibody-HER2 complexes (appearing as yellow puncta) were internalized into the cells. Consistent with the previous figures, the internalization of anti-HER2-Bs-AF647 is more rapid compared to trastuzumab-AF647 (compare D to G ). ( C , F ) Anti-HER2-Bs-AF647 and trastuzumab-AF647 mediated strikingly different trafficking of HER2 receptors at 2 h. ( C ) Internalized anti-HER2-Bs-AF647 (red) remained colocalized with HER2 receptors (green) and appeared as yellow puncta. However, trastuzumab-AF647 was dissociated from HER2 receptors and appeared as red puncta in the cells ( F ). ( G , H ) Treatment with anti-HER2-Bs, but not trastuzumab, resulted in complete surface clearance of HER2 receptors at 8 h. ( I , J ) Internalized anti-HER2-Bs-AF647 and trastuzumab-AF647 are colocalized with a lysosomal marker, LAMP1-AF488, at 8 h. The puncta of internalized anti-HER2-Bs-AF647 ( I ) or trastuzumab-AF647 ( J ) (red), colocalized with the LAMP1-AF488-positive compartments (green) merging in yellow. Treatment with anti-HER2-Bs resulted in the complete clearance of the antibody from the cell surface by 8 h ( I ). By contrast, there was a significant amount of trastuzumab remaining on the cell surface ( J ). Scale bars are 10 μm. ( K ) Treatment with anti-HER2-Bs resulted in a ~93% reduction of HER2 ectodomain (ECD) shedding in the conditioned culture media compared to the untreated cells. Treatment with trastuzumab also reduced HER2 ECD shedding by ~76%. ( L , M ) In addition, treatment with anti-HER2-Bs also reduced the number of exosome particles and the level of HER2 expression in the normalized exosomal fraction. CD63 is the protein marker for exosomes purified from conditioned culture media.

Article Snippet: Rabbit anti-human HER2 polyclonal antibody (Cat# A048529-2) was purchased from Dako, Agilent Technologies (Santa Clara, CA, USA).

Techniques: Marker, Expressing, Purification

Journal: Antibodies

Article Title: Molecular Mechanism of HER2 Rapid Internalization and Redirected Trafficking Induced by Anti-HER2 Biparatopic Antibody

doi: 10.3390/antib9030049

Figure Lengend Snippet: A proposed model of anti-HER2-Bs- and trastuzumab-mediated internalization, trafficking, and the fate of HER2 receptors. Please note that the receptors are drawn unproportionally to highlight the crosslinking on the cell surface and the intracellular trafficking. ( A ) In the untreated cells, the HER2 receptors are predominately localized on the cell surface. As part of the normal turnover process, a basal level of HER2 continuously undergoes internalization and catabolism. The pool of cell surface HER2 is replenished by the de novo synthesis of HER2 receptors. The internalized HER2 is then trafficked to multivesicular bodies (MVBs) for subsequent sorting. The HER2-loaded MVBs can fuse either with lysosomes for degradation, or with plasma membrane to release intra-luminal vesicles (ILVs) as exosomes. In addition, HER2-ECDs are constantly shedding from the receptors on the cell surface. ( B ) Treatment with trastuzumab resulted in a slow internalization of HER2 receptors via a clathrin-mediated pathway. The internalized trastuzumab-HER2 complexes are dissociated in the endosomes. The dissociated HER2 receptors are then recycled back to the cell surface. Similar to the untreated cells, a basal level of HER2 is internalized and trafficked to MVBs as a process of normal turnover, leading to either degradation of HER2 or release of HER2-loaded exosomes. On the other hand, the dissociated trastuzumab is trafficked to MVBs and lysosomes for degradation. Treatment with trastuzumab did not affect the level of exosome formation. However, binding of trastuzumab to the domain IV of the HER2 receptors on the cell surface inhibits the cleavage of HER2-ECD. ( C ) Concurrent engagement of HER2 epitopes by the bivalent bispecific anti-HER2-Bs antibody may induce crosslinking of HER2 receptors to form high molecular weight complexes on the cell surface. The anti-HER2-Bs-HER2 complexes are internalized rapidly via a clathrin-mediated pathway. The anti-HER2-Bs-HER2 complexes stay associated and traffic through the endocytic pathway. Instead of being recycled back to the cell surface, anti-HER2-Bs induces redirected trafficking of HER2 receptors to lysosomes for degradation. The degradation of HER2 receptors coincides with an increase in HER2 ubiquitination (total and K63). As a result of the lysosomal targeting, the anti-HER2-Bs-HER2-loaded multivesicular bodies are not able to fuse with the cell membrane, and thus this results in reduced exosomal export. In addition, rapid internalization of surface anti-HER2-Bs-HER2 complexes results in a fast clearance of surface HER2 receptors, thus leading to a reduction of HER2 ECD shedding on the cell surface.

Article Snippet: Rabbit anti-human HER2 polyclonal antibody (Cat# A048529-2) was purchased from Dako, Agilent Technologies (Santa Clara, CA, USA).

Techniques: Binding Assay, Molecular Weight

Journal: Cancer Biology & Therapy

Article Title: Combined targeting of EGFR and HER2 against prostate cancer stem cells

doi: 10.1080/15384047.2020.1727702

Figure Lengend Snippet: Effects of anti-EGFR and anti-HER2 monoclonal antibodies and chemotherapy treatment on prostatosphere-forming efficiency and adherent growth of DU145 cells. (a) The expression levels and the activated (phosphorylated) status of EGFR and HER2 and downstream signaling molecules, AKT, MAPK, STAT3 and SRC, were analyzed in cell line grown in adherent (Ad) and prostatosphere-forming (Sp) conditions by Western blot (one representative of two-independent experiments is shown). β-actin was used as a loading control. (b) Dose–response assay on DU145 cells treated with escalating doses of Cetuximab, Trastuzumab alone or in combination in serum-free medium and then plated under prostatosphere-forming conditions; the prostatosphere-forming efficiency was evaluated 96 h later and calculated as the ratio between the number of spheres/well and the number of single cells seeded in each well. Results (mean values±S.D.) representing one of two independent experiments, from triplicates for every treatment condition are shown. **p < .01; ***p < .001 (against control) by Bonferroni’s Multiple comparison test. Abbreviations: Ctrl = control; Cetux = Cetuximab; Trast = Trastuzumab. (c) Dose–response assay on DU145 cells treated with escalating doses of Cetuximab, Trastuzumab alone or in combination in serum-free medium under monolayer conditions. Results from MTT assay revealed no effect on cell proliferation by Cetuximab and Trastuzumab given alone or in combination. Abbreviations: Ctrl = control; Cetux = Cetuximab; Trast = Trastuzumab. (d) Cells were treated with Trastuzumab (100µg/ml), Cetuximab (25µg/ml), Taxotere (2.5nM) alone or in combination under adherent growth condition for 72 h, then trypsinized and seeded in the serum-free medium; the prostatosphere-forming efficiency was evaluated after 96 h. Results (mean values ± S.E.M) from two independent experiments, from triplicates for every treatment condition are displayed. **p< .01; *** p < .001 versus untreated cells, by Bonferroni’s Multiple comparison test. Abbreviations: Ctrl = control; Cetux = Cetuximab; Trast = Trastuzumab; Taxo = Taxotere.

Article Snippet: Formalin-fixed, paraffin-embedded tumors were sectioned at 4 µm, dewaxed, hydrated, and stained with hematoxylin and eosin or processed for IHC with: rabbit polyclonal anti-human HER2 (1:150, DAKO) after antigen retrieval with citrate buffer, pH 6.0, at 95°C for 6 min, rabbit monoclonal anti-phosphoSTAT3 (clone D3A7, 1:400, Cell Signaling Technology) after antigen retrieval with EDTA 1mM, pH 8.0 at 95°C for 15 min, with mouse monoclonal anti-EGFR (clone E30; 1:25, DAKO) upon treatment with proteinase K at 37°C for 5 min, and with mouse monoclonal anti-AR (clone AR441; 1:25, DAKO) after antigen retrieval with PTLink EDTA (DAKO) 15 min at 96°C.

Techniques: Expressing, Western Blot, MTT Assay