Journal: Cell Reports Methods

Article Title: Sequence enrichment profiles enable target-agnostic antibody generation for a broad range of antigens

doi: 10.1016/j.crmeth.2023.100475

Figure Lengend Snippet:

Article Snippet: HER2 , Sino Biological , Cat# 10004-H08H.

Techniques: Produced, Recombinant, Sequencing, Software

Journal: Biochemical pharmacology

Article Title: BRET-based assay to monitor EGFR transactivation by the AT 1 R reveals G q/11 protein-independent activation and AT 1 R-EGFR complexes

doi: 10.1016/j.bcp.2018.10.017

Figure Lengend Snippet: (A) Schematic of AT1R-EGFR BRET-based transactivation. EGFR fused to a BRET donor (Rluc8), is co-transfected with Grb2 adaptor protein tagged with a BRET acceptor (Venus) and the AT1R. Stimulation of the AT1R promotes activation of the EGFR and recruitment of Grb2. (B) HEK293 cells expressing AT1R, EGFR-Rluc8, and Grb2-Venus were treated with 10μM AngII, 1μM EGF or vehicle. Quantification of ligand-induced BRET ratio (maximum-minimum) between EGFR-Rluc8 and Grb2-Venus following AngII- and EGF-stimulation. Insert is HEK293 cells (stably expressing AT1R) stimulated with 100nM AngII, 10nM EGF or vehicle for 5 minutes before processing for phospho-ERK1/2:total-ERK1/2 (p-ERK:T-ERK) western blots. Blots are representative of 3 independent experiments (B inset) Cells expressing AT1R, Grb2-Venus and either EGFR-Rluc8, HER2-Rluc8 or HER3-Rluc8 and stimulated with 10μM AngII. Agonist stimulation is indicated by arrow. Data represent mean ± SEM of 3 independent experiments.

Article Snippet: EGFR-Rluc8, HER2-Rluc8 and HER3-Rluc8 were generated by inserting EGFR, HER2, and HER3, obtained from Origene (Rockville, MD, USA) into pcDNA3-Rluc8.

Techniques: Transfection, Activation Assay, Expressing, Stable Transfection, Western Blot

Journal: EBioMedicine

Article Title: A novel HER2-targeting antibody 5G9 identified by large-scale trastuzumab-based screening exhibits potent synergistic antitumor activity

doi: 10.1016/j.ebiom.2020.102996

Figure Lengend Snippet: Characterization of trastuzumab-optimal-synergistic-epitope (TOSE) -binding monoclonal antibodies. (a) Schematic workflow of trastuzumab-based synergistic functional screening of TOSE-binding antibodies (4H2, 4C9, 4G6, 5F12 and 5G9). 1st screening: ELISA binding and competitive ELISA with trastuzumab or pertuzumab; 2nd screening: binding to HER2-positive cells; 3rd screening: trastuzumab-based cell proliferation inhibition assay. (b) Five monoclonal antibodies (4H2, 4C9, 4G6, 5F12 and 5G9) showed trastuzumab-synergistic bioactivity higher than pertuzumab in the BT-474 cell proliferation inhibition assay. TRA: trastuzumab; PER: pertuzumab. (c) The antibody epitope grouping assay was performed by competitive ELISA. Five monoclonal antibodies (4H2, 4C9, 4G6, 5F12 and 5G9) bound to a unique epitope-TOSE, which was different from both the trastuzumab-binding epitope and pertuzumab-binding epitope. (d) The binding specificity and species cross-reactivity were determined by ELISA. 5G9 bound to HER2 protein, but not to other EGFR family members, including EGFR, HER3 and HER4. (e) The cross-species binding reactivity was determined by ELISA. 5G9 was able to bind cynomolgus HER2 protein but not murine HER2 protein. The BT-474 cell proliferation inhibition assay, antibody epitope grouping assay and the cross-reactivity assay were repeated three times and the results were shown as the mean ± SD ( n = 3). Two-way ANOVA, *** P< 0.001.

Article Snippet: The recombinant proteins huEGFR (Cat# 10001-H02H), huHER2 (Cat# 10004-H02H), huHER3 (Cat# 10201-H02H), huHER4 (Cat# 10363-H02H), rhesus HER2 (Cat# 90020-K02H) and mouse HER2 (Cat# 50714-M02H) were purchased from Sino Biological Inc.

Techniques: Binding Assay, Functional Assay, Enzyme-linked Immunosorbent Assay, Competitive ELISA, Inhibition

Journal: EBioMedicine

Article Title: A novel HER2-targeting antibody 5G9 identified by large-scale trastuzumab-based screening exhibits potent synergistic antitumor activity

doi: 10.1016/j.ebiom.2020.102996

Figure Lengend Snippet: 5G9 and trastuzumab synergistically inhibit HER2-positive cancer cell growth. (a) Inhibition of BT-474 cell viability was examined by Cell Titer Glo after 6 days of antibody treatment. The initial antibody concentration was 100 nM for both the monoclonal antibody and the antibody combination (1:1 ratio). Growth inhibition was calculated as the fraction of viable cells in the antibody treated group compared with untreated control cells. The cell proliferation inhibition assay was repeated three times and the results were shown as the mean ± SD ( n = 3). Two-way ANOVA, ** P< 0.01. TRA: trastuzumab; PER: pertuzumab. (b) Dose-effect plots and combination index (CI.) plots of the two combinations were calculated by using the method of Chou and Talalay with the commercial software Calcusyn. C.I. values for effective doses at which 50%, 75%, and 90% (ED 50 , ED 75 , and ED 90 , respectively) of cells were killed. Drug synergy was defined by C.I. values less than 1, the lower C.I. value, the more synergy. (c) Representative images from the EdU assay of BT-474 cells. The viable cells were stained blue by DAPI (blue) and the proliferation cells were stained red by EdU (red).The EdU assay was repeated three times and the representative images were shown. (d) Human breast cancer cell lines (SK-BR-3, AU-565, MCF-7) and human gastric cell lines (NCI-N87) were treated with 100 nM antibodies for 3 or 6 days and cell viability was determined by Cell Titer Glo. The experiments were repeated three times and the cell viability is shown as the mean ± SD ( n = 3). The relative HER2 expression of cell lines was detected and analyzed by flow cytometry and FlowJo software.

Article Snippet: The recombinant proteins huEGFR (Cat# 10001-H02H), huHER2 (Cat# 10004-H02H), huHER3 (Cat# 10201-H02H), huHER4 (Cat# 10363-H02H), rhesus HER2 (Cat# 90020-K02H) and mouse HER2 (Cat# 50714-M02H) were purchased from Sino Biological Inc.

Techniques: Inhibition, Concentration Assay, Software, EdU Assay, Staining, Expressing, Flow Cytometry

Journal: EBioMedicine

Article Title: A novel HER2-targeting antibody 5G9 identified by large-scale trastuzumab-based screening exhibits potent synergistic antitumor activity

doi: 10.1016/j.ebiom.2020.102996

Figure Lengend Snippet: 5G9 and trastuzumab enhance cell apoptosis, HER2 internalization and disruption. (a) BT-474 cells were treated with 100 nM antibodies for 3 days. The number of viable cells was detected by Trypan Blue, and annexin-V and PI were used to stain early apoptotic cells and dead cells. TRA: trastuzumab; PER: pertuzumab. (b) BT-474 cells were treated with 100 nM antibodies for 3 days, and cell lysates were obtained and immunoblotted with a poly (ADP-ribose) polymerase (PARP) polyclonal antibody. Cleaved PARP is represented by 89 KD-fragments and full-length PARP (116 KD). (c) HER2 internalization induced by 5G9, trastuzumab or pertuzumab in SK-BR-3 cells. (d) The effect of trastuzumab on the internalization of 5G9 or pertuzumab in SK-BR-3 cells. FITClabeled 5G9 or pertuzumab was detected and analyzed by flow cytometry and FlowJo software. (e) The effect of 5G9 or pertuzumab on the internalization of trastuzumab in SK-BR-3 cells. APC labeled trastuzumab was detected and analyzed by flow cytometry and FlowJo software. (f) Effect of HER2 single agents or combination on receptor phosphorylation and downstream signaling cascade. BT-474 cells were treated with 100 nM antibodies for 3 days, and cell lysates were obtained and immunoblotted with antibodies against EGFR, p-EGFR, HER2, p-HER2, HER3, p-HER3, AKT, p-AKT, ERK, p-ERK, and GAPDH. All experiments were repeated three times and the representative images and data were shown. Two-way ANOVA, * P< 0.05, *** P< 0.001.

Article Snippet: The recombinant proteins huEGFR (Cat# 10001-H02H), huHER2 (Cat# 10004-H02H), huHER3 (Cat# 10201-H02H), huHER4 (Cat# 10363-H02H), rhesus HER2 (Cat# 90020-K02H) and mouse HER2 (Cat# 50714-M02H) were purchased from Sino Biological Inc.

Techniques: Staining, Flow Cytometry, Software, Labeling

Journal: Experimental and Therapeutic Medicine

Article Title: IL-35 improves T reg -mediated immune suppression in atherosclerotic mice

doi: 10.3892/etm.2016.3649

Figure Lengend Snippet: Plasma levels of Foxp3. Peripheral blood mononuclear cells were collected from each group, and the proportions of CD4 + CD25 + Foxp3 + T reg /CD4 + T-cells were analyzed by flow cytometry, and quantified using FlowJo 7.6.1 software. (A) Histogram. Data are presented as the mean ± standard deviation. *P<0.01, vs. the AS group. (B) Negative group, (C) AS group, (D) atorvastatin group and (E) IL-35 group. Foxp3, forkhead box protein 3; AS, atherosclerosis; IL-35, interleukin-35; APC, allophycocyanin; PE, phycoerythrin.

Article Snippet: Anti-Foxp3 antibody was purchased from Wuhan Boster Biological Technology, Ltd. (Wuhan, China).

Techniques: Clinical Proteomics, Flow Cytometry, Software, Standard Deviation

Journal: Experimental and Therapeutic Medicine

Article Title: IL-35 improves T reg -mediated immune suppression in atherosclerotic mice

doi: 10.3892/etm.2016.3649

Figure Lengend Snippet: Levels of Foxp3 in atherosclerotic lesions. The deposition of Foxp3 in arteries from various groups was detected by immunohistochemistry (magnification, ×400). Positive Foxp3 was shown as brown nuclei. In the (A) negative and (B) AS groups, the deposition of Foxp3 in the lesions was minimal. Conversely, Foxp3 deposition was increased in the lesions of the (C) atorvastatin and (D) IL-35 groups, as compared with the AS group. (E) This was shown to be significant following quantification. There was no significant difference in the levels of Foxp3 between the atorvastatin and IL-35 groups. *P<0.01, vs. the negative control. Foxp3, forkhead box protein 3; AS, atherosclerosis; IL-35, interleukin-35.

Article Snippet: Anti-Foxp3 antibody was purchased from Wuhan Boster Biological Technology, Ltd. (Wuhan, China).

Techniques: Immunohistochemistry, Negative Control