rabbit anti human phospho her2 erbb2 tyr1221 1222  (Cell Signaling Technology Inc)

Journal: Cancers

Article Title: Characterization of HER2-Positive Murine Breast Cancer Models for Investigating HER2-Targeted Therapy and Immunotherapy

doi: 10.3390/cancers18060997

Figure Lengend Snippet: Validation of HER2 expression in the newly established HER2-positive mouse tumors. ( A ) Western blotting analysis of HER2 and phosphor-HER2 in transduced 4T1 and EO771 cell lines. BT474 included as HER2+ control. ( B ) Representative immunofluorescence staining of HER2 expression in transduced EO771 cells. ( C ) Tumor volume curve of 4T1 ( top ) and EO771 ( bottom ) HER2-OE lines (n = 4–5 per group). Data is mean ± SD. ( D ) Representative immunohistochemistry staining of HER2 expression on tumor slices. The uncropped blots are included in .

Article Snippet: Briefly, for IF, cells grown in 96-well plates were fixed with 10% formalin for 30 min at room temperature and blocked with 1% BSA in PBS for 1 h. Cells were incubated overnight at 4 °C with primary rabbit anti-human HER2 antibody (Cell Signaling Technology, 2242S, 1:100 dilution), followed by incubation with Cy5-conjugated goat anti-rabbit secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA, 711-175-152, 1:200) for 1 h at room temperature in the dark.

Techniques: Biomarker Discovery, Expressing, Western Blot, Control, Immunofluorescence, Staining, Immunohistochemistry

Journal: Cancers

Article Title: Characterization of HER2-Positive Murine Breast Cancer Models for Investigating HER2-Targeted Therapy and Immunotherapy

doi: 10.3390/cancers18060997

Figure Lengend Snippet: Trastuzumab alone was not able to reduce primary tumor size but decreased tumor brain metastasis in 4T1-HER2 models. ( A ) Tumor volume curve of 4T1-GFP, -HER2 WT , and -HER2 YVMA tumors treated with either saline or trastuzumab (15 mg/kg, IV). Filled shapes represent saline-treated groups; open shapes represent trastuzumab-treated groups. No statistically significant difference was observed between groups (repeated measures ANOVA p = 0.56). ( B ) Plot of average radiance from GFP fluorescence in the brains of 4T1 tumor-bearing mice. Symbols represent individual mice. ( C ) Fluorescence images of the brains of 4T1 tumor-bearing mice. TRA, trastuzumab. Data is shown as mean ± SD. Two-way ANOVA with Tukey’s HSD test was used in ( B ). *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: Briefly, for IF, cells grown in 96-well plates were fixed with 10% formalin for 30 min at room temperature and blocked with 1% BSA in PBS for 1 h. Cells were incubated overnight at 4 °C with primary rabbit anti-human HER2 antibody (Cell Signaling Technology, 2242S, 1:100 dilution), followed by incubation with Cy5-conjugated goat anti-rabbit secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA, 711-175-152, 1:200) for 1 h at room temperature in the dark.

Techniques: Saline, Fluorescence

Journal: Cancers

Article Title: Characterization of HER2-Positive Murine Breast Cancer Models for Investigating HER2-Targeted Therapy and Immunotherapy

doi: 10.3390/cancers18060997

Figure Lengend Snippet: HER2-targeted therapy synergizes with immunotherapy in HER2-overexpressing (OE) tumors. ( A ) Tumor volume ( left ) and survival ( right ) curves for EO771-HER2 YVMA and 4T1-HER2 YVMA models treated with saline, anti-HER2 agents (TRA + TUC), ICB (αPD-1 and αCTLA-4), or combination therapy (anti-HER2 + ICB) as indicated by arrows. ( B , C ) Flow cytometry quantification of CD4+ ( B ) and CD8+ ( C ) T cells in EO771-HER2 YVMA tumors on day 5. Mantel–Cox logrank test was used for survival analysis in ( A ). Two-way ANOVA with Bonferroni correction for multiple comparisons was used for ( B , C ). All data are shown as mean ± SD; * p < 0.05; ** p < 0.01.

Article Snippet: Briefly, for IF, cells grown in 96-well plates were fixed with 10% formalin for 30 min at room temperature and blocked with 1% BSA in PBS for 1 h. Cells were incubated overnight at 4 °C with primary rabbit anti-human HER2 antibody (Cell Signaling Technology, 2242S, 1:100 dilution), followed by incubation with Cy5-conjugated goat anti-rabbit secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA, 711-175-152, 1:200) for 1 h at room temperature in the dark.

Techniques: Saline, Flow Cytometry

Journal: Cancers

Article Title: Characterization of HER2-Positive Murine Breast Cancer Models for Investigating HER2-Targeted Therapy and Immunotherapy

doi: 10.3390/cancers18060997

Figure Lengend Snippet: HER2-overexpressing models show differential responses to T-DM1 and T-Dxd. Tumor volume curves for BT474 (human HER2+) ( A ), HER2− MDA-MB-231 (human TNBC) ( B ), 4T1-HER2 WT ( C ), and EO771-HER2 WT ( D ) treated with saline (black line), T-DM1 (blue line), or T-Dxd (red line). n = 3 per group. * p < 0.05.

Article Snippet: Briefly, for IF, cells grown in 96-well plates were fixed with 10% formalin for 30 min at room temperature and blocked with 1% BSA in PBS for 1 h. Cells were incubated overnight at 4 °C with primary rabbit anti-human HER2 antibody (Cell Signaling Technology, 2242S, 1:100 dilution), followed by incubation with Cy5-conjugated goat anti-rabbit secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA, 711-175-152, 1:200) for 1 h at room temperature in the dark.

Techniques: Saline

Journal: Journal of the American Chemical Society

Article Title: Hijacking Extracellular Targeted Protein Degrader–Drug Conjugates for Enhanced Drug Delivery

doi: 10.1021/jacs.5c15047

Figure Lengend Snippet: LIPTAC-mediated degradation of multiple extracellular proteins. (a,b) Schematic illustration of PD-L1 targeting LIPTAC, and Western blot showing total degradation of PD-L1 on MDA-MB-231 cells following 24 h treatment of 50 nM monomeric anti-PD-L1 parent, Atz, or LIPTAC containing Atz. Percent PD-L1 levels were quantified by ImageJ relative to PBS control. Each sample was tested in biological triplicate and error bars represent standard deviations. Statistics were calculated by unpaired two-tailed student t test. * P < 0.05. ** P < 0.01. (c) Western blot analysis showing lysosome-dependent PD-L1 degradation on MDA-MB-231 cells. Cells were pretreated with either 500 nM Bafilomycin A (BafA) or 500 nM MG132 for 1 h followed by 24 h treatment with 50 nM LIPTAC. (d) Schematic illustration of HER2-targeting LIPTAC and Western blot analysis showing total HER2 degradation on MCF7 cells following 24 h treatment of monomeric Traz, LIPTAC and KineTAC. Percent ERBB2 (HER2) levels were quantified by ImageJ relative to PBS control. Data represent at least two independent experiments. (e) Schematic illustration of CXCR4-targeting LIPTAC and Western blot analysis showing total CXCR4 degradation on HeLa cells following 24 h treatment of Nb monomer, monomeric 142F1, or LIPTAC. (f,g) Schematic illustration of cleaved CDCP1 (cCDCP1)-targeting LIPTAC and degradation of CDCP1 in PL45 cells following 24 h treatment of 50 nM cCDCP1 binder CL03 IgG, full-length CDCP1 (flCDCP1) binder 4A06 IgG, or cCDCP1-specific LIPTAC. Each sample was tested in biological triplicate and error bars represent standard deviations. Statistics were calculated by unpaired two-tailed student t test. * P < 0.05. ** P < 0.01. ns, not significant.

Article Snippet: Antibodies used included rabbit anti-human EGFR (Cell Signaling Technology, catalog no. 4267S, 1:1000), rabbit anti-human PD-L1 (Cell Signaling Technology, catalog no. 13684S, 1:1000), rabbit anti-human CXCR4 (Cell Signaling Technology, catalog no. 64837S, 1:1000), rabbit anti-human ERBB2 (Cell Signaling Technology, catalog no. 4290S, 1:1000), rabbit anti-human CDCP1 (Cell Signaling Technology, catalog no. 13794S, 1:1000), mouse anti-human α-tubulin (Cell Signaling Technology, 3873S, 1:3000), goat anti-human LDLR (R&D Systems, catalog no. AF2148, 1:1000), IRDye 800CW goat anti-rabbit IgG (LI-COR Biosciences, catalog no. 926-32211), IRDye 680RD goat anti-mouse IgG (LI-COR Biosciences, catalog no. 926-68070, 1:5000), IRDye 800CW donkey anti-goat IgG (LI-COR Biosciences, catalog no. 926-32214, 1:5000), and peroxidase goat anti-rabbit IgG (H+L) (Jackson ImmunoResearch, catalog no. 111-035-144, 1:5000).

Techniques: Western Blot, Control, Two Tailed Test