Journal: British Journal of Cancer

Article Title: Targeting ErbB2 and ErbB3 with a bispecific single-chain Fv enhances targeting selectivity and induces a therapeutic effect in vitro

doi: 10.1038/sj.bjc.6604700

Figure Lengend Snippet: Expression levels of ErbB2 and ErbB3 on cell lines

Article Snippet: Anti-ErbB2 (Becton Dickinson, cat no. 340552) and anti-ErbB3 (R&D, cat no. FAB3481P) conjugated to a 1 : 1 ratio with phycoerythrin (PE) by the manufacturers were used for monitoring expression levels with the mean of three independent experiments reported in .

Techniques: Expressing

Journal: British Journal of Cancer

Article Title: Targeting ErbB2 and ErbB3 with a bispecific single-chain Fv enhances targeting selectivity and induces a therapeutic effect in vitro

doi: 10.1038/sj.bjc.6604700

Figure Lengend Snippet: Affinity and binding kinetics of the anti-ErbB3 A5 scFv. k on and k off rates were determined by surface plasmon resonance and used to determine the binding affinity ( K D ) of the A5 scFv. ( A ) Sensorgram fit to 1 : 1 Langmuir binding model. ( B ) Analysis of data.

Article Snippet: Anti-ErbB2 (Becton Dickinson, cat no. 340552) and anti-ErbB3 (R&D, cat no. FAB3481P) conjugated to a 1 : 1 ratio with phycoerythrin (PE) by the manufacturers were used for monitoring expression levels with the mean of three independent experiments reported in .

Techniques: Binding Assay, SPR Assay

Journal: British Journal of Cancer

Article Title: Targeting ErbB2 and ErbB3 with a bispecific single-chain Fv enhances targeting selectivity and induces a therapeutic effect in vitro

doi: 10.1038/sj.bjc.6604700

Figure Lengend Snippet: The anti-ErbB2/ErbB3 bs-scFv ALM. ( A ) Cartoon of ALM depicting scFv orientation, linker sequence and kinetic constants of ALM for each target antigen. ( B ) UV adsorption spectrum chromatograph of ALM over Superdex 75 size-exclusion column.

Article Snippet: Anti-ErbB2 (Becton Dickinson, cat no. 340552) and anti-ErbB3 (R&D, cat no. FAB3481P) conjugated to a 1 : 1 ratio with phycoerythrin (PE) by the manufacturers were used for monitoring expression levels with the mean of three independent experiments reported in .

Techniques: Sequencing, Adsorption

Journal: British Journal of Cancer

Article Title: Targeting ErbB2 and ErbB3 with a bispecific single-chain Fv enhances targeting selectivity and induces a therapeutic effect in vitro

doi: 10.1038/sj.bjc.6604700

Figure Lengend Snippet: ALM selectively targets ErbB2/ErbB3 positive cells in vitro

Article Snippet: Anti-ErbB2 (Becton Dickinson, cat no. 340552) and anti-ErbB3 (R&D, cat no. FAB3481P) conjugated to a 1 : 1 ratio with phycoerythrin (PE) by the manufacturers were used for monitoring expression levels with the mean of three independent experiments reported in .

Techniques: In Vitro

Journal: British Journal of Cancer

Article Title: Targeting ErbB2 and ErbB3 with a bispecific single-chain Fv enhances targeting selectivity and induces a therapeutic effect in vitro

doi: 10.1038/sj.bjc.6604700

Figure Lengend Snippet: The A5-linker-ML3.9 bs-scFv selectively binds BT-474 tumour cells in vitro . Non-labelled BT-474 (ErbB2‘+’/ErbB3‘+’) breast tumour cells were mixed with either an equal ( A and B ) or 18-fold excess ( C ) of fluorescently labelled MCF10a (ErbB2‘±’/ErbB3‘±’) normal breast epithelial cells. Cell mixtures were then incubated with buffer ( A ) or 100 n M ALM ( B and C ) and binding of ALM to each cell population was determined by flow cytometry with an anti-6XHis tag secondary antibody. MCF10a cells were sorted to the upper quadrants and the non-labelled BT-474 cells were sorted to the lower quadrants. Cells bound by the secondary antibody sorted to the respective right hand quadrants. Images on the left depict the raw flow cytometry data. Values on the right represent the absolute number and overall percentage of each cell type in the respective quadrants.

Article Snippet: Anti-ErbB2 (Becton Dickinson, cat no. 340552) and anti-ErbB3 (R&D, cat no. FAB3481P) conjugated to a 1 : 1 ratio with phycoerythrin (PE) by the manufacturers were used for monitoring expression levels with the mean of three independent experiments reported in .

Techniques: In Vitro, Incubation, Binding Assay, Flow Cytometry

Journal: British Journal of Cancer

Article Title: Targeting ErbB2 and ErbB3 with a bispecific single-chain Fv enhances targeting selectivity and induces a therapeutic effect in vitro

doi: 10.1038/sj.bjc.6604700

Figure Lengend Snippet: Bispecific binding is required for optimal tumour targeting of the ALM bs-scFv in vivo . The biodistributions of radioiodinated ALM, ALD and DLM bs-scFv were analysed 24 h post-injection into xenograft-bearing SCID mice ( n =5 per cohort). ( A ) Co-expression of ErbB2 and ErbB3 by the targeted tumour is required for optimal targeting of ALM in vivo . 125 I-ALM targeted ErbB2+/ErbB3+ tumour xenografts to ⩾3-fold higher levels than xenografts that express only one of the target antigens. ( B ) Radioiodinated ALM ( 125 I-ALM), which is capable of bivalent association with the surface of Sk-OV-3 tumour cells, exhibited increased targeting as compared with ALD and DLM that targeted the tumours monovalently. Error bars represent the standard error of the mean (s.e.m.).

Article Snippet: Anti-ErbB2 (Becton Dickinson, cat no. 340552) and anti-ErbB3 (R&D, cat no. FAB3481P) conjugated to a 1 : 1 ratio with phycoerythrin (PE) by the manufacturers were used for monitoring expression levels with the mean of three independent experiments reported in .

Techniques: Binding Assay, In Vivo, Injection, Expressing

Journal: British Journal of Cancer

Article Title: Targeting ErbB2 and ErbB3 with a bispecific single-chain Fv enhances targeting selectivity and induces a therapeutic effect in vitro

doi: 10.1038/sj.bjc.6604700

Figure Lengend Snippet: The A5-linker-ML3.9 bs-scFv has intrinsic anti-tumour cell activity. ( A ) Treatment of BT-474 and MDA-361/DYT2 cells with ALM inhibits colony formation in clonogenicity assays. Treatment of ( B ) BT-474 or ( C ) MDA-361/DYT2 cells with A5 scFv, ML3.9 scFv or the combination of both indicates that the majority of the intrinsic anti-tumour cell activity of ALM is due to the anti-ErbB3 A5 scFv arm. Colonies larger than 0.35 m M were counted using an automatic colony counter. Error bars represent the standard deviation.

Article Snippet: Anti-ErbB2 (Becton Dickinson, cat no. 340552) and anti-ErbB3 (R&D, cat no. FAB3481P) conjugated to a 1 : 1 ratio with phycoerythrin (PE) by the manufacturers were used for monitoring expression levels with the mean of three independent experiments reported in .

Techniques: Activity Assay, Standard Deviation

Journal: Oncotarget

Article Title: Tumor suppressor role of miR-3622b-5p in ERBB2-positive cancer.

doi: 10.18632/oncotarget.14968

Figure Lengend Snippet: Figure 1: ERBB2 as target of miR-3622-5p. A. Western blot analysis showing suppressed ERBB2 protein levels in SK-BR-3 breast cancer cells and SNU-216 gastric cancer cells after miR-3622-5p over-expression. The over-expression of miR-3622-5p does not alter the expression level of ERBB3. GAPDH as the internal control. Graphical presentation in the right panel. B. The seed sequence of miR-3622b-5p is complementary to the 3’-UTR of ERBB2. C. Luciferase assay showing reduction in reporter activity (relative luciferase units) after co-transfection of wild type ERBB2 3’-UTR (ERBB2-3’UTR-WT) or the fragment of ERBB2 3’-UTR lacking the candidate miR-3622b-5p binding sequence (ERBB2-3’-UTR-del) with miR-3622-5p into HEK-293T cells. ns, no significance. D. Luciferase assay showing reduction in reporter activity after transfection of ERBB2-3’UTR-WT or ERBB2-3’-UTR-del in MCF-10A cells. E. Luciferase assay showing reduction in reporter activity after transfection of ERBB2-3’UTR-WT or ERBB2-3’-UTR-del in SK-BR-3 cells.

Article Snippet: The primary antibody for ERBB3 was purchased from Proteintech Wuhan Sanying (Wuhan, China).

Techniques: Western Blot, Over Expression, Expressing, Control, Sequencing, Luciferase, Activity Assay, Cotransfection, Binding Assay, Transfection

Journal: International Journal of Molecular Sciences

Article Title: The Effect of Valine on the Synthesis of α-Casein in MAC-T Cells and the Expression and Phosphorylation of Genes Related to the mTOR Signaling Pathway

doi: 10.3390/ijms26073179

Figure Lengend Snippet: Effects of different valine concentrations on the phosphorylation of downstream proteins in the mTOR signaling pathway. ( A ) Western blotting was used to detect the protein expression levels of phosphorylated mTOR (P-mTOR) and phosphorylated S6K1 (P-S6K1). ( B ) The phosphorylation level of mTOR protein (P-mTOR/mTOR) was quantified and calculated using β-actin and mTOR as internal references. ( C ) The phosphorylation level of S6K1 protein (P-S6K1/S6K1) was quantified and calculated using β-actin and S6K1 as internal references. ( D ) Western blotting was used to detect the protein expression levels of phosphorylated 4EBP1 (P-4EBP1) and phosphorylated RPS6 (P-RPS6). ( E ) The phosphorylation level of 4EBP1 protein (P-4EBP1/4EBP1) was quantified and calculated using β-actin and 4EBP1 as internal references. ( F ) The phosphorylation level of RPS6 protein (P-RPS6/RPS6) was quantified and calculated using β-actin and RPS6 as internal references. ( G ) Western blotting was used to detect the protein expression levels of phosphorylated eEF2 (P-eEF2). ( H ) The phosphorylation level of eEF2 protein (P-eEF2/eEF2) was quantified and calculated using β-actin and eEF2 as internal references. The error bars represent the SD ( n = 3). ANOVA was used to detect the differences between groups, and the LSD method was combined for uncorrected exploratory pairwise comparisons, while the Duncan method was used for conservative multiple comparison corrections. Different lowercase letters indicate significant differences ( p < 0.05), while different uppercase letters indicate significant differences ( p < 0.01).

Article Snippet: , RPS6 , biorbyt , orb585017 , 5% egg white , 1:1000.

Techniques: Phospho-proteomics, Western Blot, Expressing, Comparison

Journal: International Journal of Molecular Sciences

Article Title: The Effect of Valine on the Synthesis of α-Casein in MAC-T Cells and the Expression and Phosphorylation of Genes Related to the mTOR Signaling Pathway

doi: 10.3390/ijms26073179

Figure Lengend Snippet: Effects of valine and rapamycin on the phosphorylation levels of mTOR downstream proteins. ( A ) The phosphorylation levels of mTOR (P-mTOR) in MAC-T cells treated with varying concentrations of rapamycin were assessed using Western blotting. ( B ) The P-mTOR level (P-mTOR/mTOR) in MAC-T cells treated with 100 nM rapamycin was quantified and calculated using β-actin and mTOR as internal references. ( C ) Western blotting was used to detect the protein expression levels of P-mTOR and P-S6K1. ( D ) The P-mTOR level (P-mTOR/mTOR) was quantified and calculated using β-actin and mTOR as internal references. ( E ) The P-S6K1 level (P-S6K1/S6K1) was quantified and calculated using β-actin and S6K1 as internal references. ( F ) Western blotting was used to detect the protein expression levels of P-4EBP1 and P-RPS6. ( G ) The P-4EBP1 level (P-4EBP1/4EBP1) was quantified and calculated using β-actin and 4EBP1 as internal references. ( H ) The P-RPS6 level (P-RPS6/RPS6) was quantified and calculated using β-actin and RPS6 as internal references. ( I ) Western blotting was used to detect the protein expression levels of P-eEF2 and α-casein. ( J ) The P-eEF2 level (P-eEF2/eEF2) was quantified and calculated using β-actin and eEF2 as internal references. ( K ) The α-casein protein expression level was quantified using β-actin as an internal reference. The error bars represent the SD ( n = 3). ANOVA was used to detect the differences between groups, and the LSD method was combined for uncorrected exploratory pairwise comparisons, while the Duncan method was used for conservative multiple comparison corrections. Different lowercase letters indicate significant differences ( p < 0.05), while different uppercase letters indicate significant differences ( p < 0.01).

Article Snippet: , RPS6 , biorbyt , orb585017 , 5% egg white , 1:1000.

Techniques: Phospho-proteomics, Western Blot, Expressing, Comparison

Journal: International Journal of Molecular Sciences

Article Title: The Effect of Valine on the Synthesis of α-Casein in MAC-T Cells and the Expression and Phosphorylation of Genes Related to the mTOR Signaling Pathway

doi: 10.3390/ijms26073179

Figure Lengend Snippet: Primer sequences of internal reference genes and target genes.

Article Snippet: , RPS6 , biorbyt , orb585017 , 5% egg white , 1:1000.

Techniques: Sequencing

Journal: International Journal of Molecular Sciences

Article Title: The Effect of Valine on the Synthesis of α-Casein in MAC-T Cells and the Expression and Phosphorylation of Genes Related to the mTOR Signaling Pathway

doi: 10.3390/ijms26073179

Figure Lengend Snippet: Detailed information of antibodies.

Article Snippet: , RPS6 , biorbyt , orb585017 , 5% egg white , 1:1000.

Techniques: