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: 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

Journal: PLoS ONE

Article Title: Engineering of Bispecific Affinity Proteins with High Affinity for ERBB2 and Adaptable Binding to Albumin

doi: 10.1371/journal.pone.0103094

Figure Lengend Snippet: Top: Sequence of ADAPT ERBB2-1 and ten representative candidates found after phage display affinity maturation compared to ABD before randomization. One sequence (ADAPT ERBB2-mat10 ) contains a substitution of a scaffold residue that was not intentionally diversified in the library. The frequency of each clone in the sequence data (among 438 sequences) and tracks (A–H) where the corresponding sequence was found are indicated to the right. Track D was underrepresented in the sequence data set. Bottom: Sequences of 21 clones identified after rounds three and four of FACS sorting. The total frequency in the sequence data (278 sequences) is shown and the two numbers in brackets indicate the number of times each sequence was observed in selections without or with HSA present, respectively. Nine of the variants contained scaffold substitutions and modifications in the region spanning helix two and three were common in sequences observed after selections in the presence of HSA. 13 of these candidates were also observed after two rounds of FACS (not shown) and two of them had been observed already among the clones obtained after four rounds of phage display (identical sequences are indicated by asterisks).

Article Snippet: ERBB2-Fc chimera (R&D Systems) was used as target in half of the tracks (C, D, G and H) and biotinylated (using Biotin-XX-succinimidyl ester B-1606, Invitrogen) ERBB2-His 6 (Sino Biological) was used in the remaining tracks (A, B, E and F).

Techniques: Sequencing, Clone Assay

Journal: PLoS ONE

Article Title: Engineering of Bispecific Affinity Proteins with High Affinity for ERBB2 and Adaptable Binding to Albumin

doi: 10.1371/journal.pone.0103094

Figure Lengend Snippet: Affinities and melting temperatures (T m ) for the non-randomized albumin-binding domain (ABD), ADAPT ERBB2-1 and variants of ADAPT ERBB2-1 .

Article Snippet: ERBB2-Fc chimera (R&D Systems) was used as target in half of the tracks (C, D, G and H) and biotinylated (using Biotin-XX-succinimidyl ester B-1606, Invitrogen) ERBB2-His 6 (Sino Biological) was used in the remaining tracks (A, B, E and F).

Techniques: Binding Assay

Journal: PLoS ONE

Article Title: Engineering of Bispecific Affinity Proteins with High Affinity for ERBB2 and Adaptable Binding to Albumin

doi: 10.1371/journal.pone.0103094

Figure Lengend Snippet: ( A ). ERBB2-binding with and without an excess of HSA. Cells expressing variants from tracks selected without (A, C, E and G) or with (B, D, F and H) 1 µM HSA present were incubated with 50 nM ERBB2 +/− 1 µM HSA and analyzed by flow-cytometry. ERBB2-binding was detected through incubation with streptavidin-R-phycoerythrin (y-axis) and surface expression levels by IgG-Alexa Fluor 647 conjugate (x-axis), data are shown using logarithmic scales. Each diagram presents an overlay of contour plots of the expressing populations of cells from two separate samples from the same batch (A-H) incubated with only ERBB2 (blue) or ERBB2 and HSA (red). The contours represent cell density in the respective regions corresponding to 20, 40, 60 or 80% of the maximum cell density observed. Each sample is represented by 25–30000 events. Corresponding data for ADAPT ERBB2-1 and scaffold ABD are shown in the inlay of panel A to facilitate a comparison. The library each track is derived from, the form of ERBB2 and concentrations used during phage display selection are also indicated. (B ). HSA-binding. Cells from tracks selected without (A, C, E and G) or with (B, D, F and H) 1 µM HSA present were incubated with 50 nM HSA-Alexa Fluor 488 conjugate and analyzed by flow-cytometry. HSA-binding is shown on the y-axis and surface expression level, detected by IgG-Alexa Fluor 647 conjugate, on the x-axis. As controls, cells expressing ADAPT ERBB2-1 or scaffold ABD are included.

Article Snippet: ERBB2-Fc chimera (R&D Systems) was used as target in half of the tracks (C, D, G and H) and biotinylated (using Biotin-XX-succinimidyl ester B-1606, Invitrogen) ERBB2-His 6 (Sino Biological) was used in the remaining tracks (A, B, E and F).

Techniques: Binding Assay, Expressing, Incubation, Flow Cytometry, Derivative Assay, Selection

Journal: PLoS ONE

Article Title: Engineering of Bispecific Affinity Proteins with High Affinity for ERBB2 and Adaptable Binding to Albumin

doi: 10.1371/journal.pone.0103094

Figure Lengend Snippet: 21 clones identified after rounds three and four of FACS sorting were screened for ERBB2-binding ( A ), HSA-binding ( B ) and ERBB2-binding in the presence of an excess of HSA ( C ). 5 nM ERBB2 was used in screen A and the binding signal divided by the surface expression level for each clone was normalized against the signal from ADAPT ERBB2-1 . 20 nM HSA was used in screen B and the signals were normalized to scaffold ABD. 5 nM ERBB2 together with 1 µM unlabeled HSA was used in screen C and ADAPT ERBB2-1 was used for normalization. All measurements were done in duplicate on different days and the bars indicate the standard deviation.

Article Snippet: ERBB2-Fc chimera (R&D Systems) was used as target in half of the tracks (C, D, G and H) and biotinylated (using Biotin-XX-succinimidyl ester B-1606, Invitrogen) ERBB2-His 6 (Sino Biological) was used in the remaining tracks (A, B, E and F).

Techniques: Clone Assay, Binding Assay, Expressing, Standard Deviation

Journal: PLoS ONE

Article Title: Engineering of Bispecific Affinity Proteins with High Affinity for ERBB2 and Adaptable Binding to Albumin

doi: 10.1371/journal.pone.0103094

Figure Lengend Snippet: Affinities of selected affinity matured ADAPTs after FACS.

Article Snippet: ERBB2-Fc chimera (R&D Systems) was used as target in half of the tracks (C, D, G and H) and biotinylated (using Biotin-XX-succinimidyl ester B-1606, Invitrogen) ERBB2-His 6 (Sino Biological) was used in the remaining tracks (A, B, E and F).

Techniques: Binding Assay

Journal: PLoS ONE

Article Title: Engineering of Bispecific Affinity Proteins with High Affinity for ERBB2 and Adaptable Binding to Albumin

doi: 10.1371/journal.pone.0103094

Figure Lengend Snippet: ( A ). ERBB2-binding at high concentrations of HSA. Four variants of ADAPT ERBB2-FACS-12 (A8) were displayed at the bacterial surface and incubated with labeled ERBB2 (10 nM) in the presence of varying concentrations of HSA. All mutated variants show a retained ability to bind ERBB2 at very high concentrations of albumin, which is in contrast to the parental ADAPT ERBB2-FACS-12 (A8) . Increasing streptavidin-R-phycoerythrin signals were observed at high concentrations of HSA. This effect was only seen for molecules with retained ERBB2-binding and was also observed for an ERBB2-binding Affibody molecule used as control . Hence, this experimental artifact was not ADAPT-specific. All measurements were done in duplicate on different days and the bars indicate the standard deviation. The albumin concentration is shown on a logarithmic scale, the two highest concentrations were 300 and 600 µM, respectively. ( B ). Competition experiment using ERBB2-binding proteins expressed on staphylococcal cells and flow cytometry. ERBB2-binding (5 nM) to cell-displayed binders was always blocked by a 10-fold excess of the same protein in a soluble form. Only ADAPTs and trastuzumab competed with each other for ERBB2-binding, no competition with the ERBB2-binding Affibody molecule Z ERBB2 was observed. All measurements were done in duplicate on different days and the bars indicate the standard deviation. ( C ). Binding to native ERBB2 on SKOV-3 cells. Cells were incubated with 100 nM biotinylated ADAPT ERBB2-1 or ADAPT ERBB2-FACS-12 (A8) and detected with streptavidin-R-phycoerythrin conjugate. Pre-incubating the cells with an excess of either non-labeled ADAPT ERBB2-FACS-12 (A8) or trastuzumab scFv blocked binding of ADAPT ERBB2-FACS-12 (A8) . A two-sample two-tailed t-test demonstrated that the binding for ADAPT ERBB2-1 was significantly stronger than the signal from the scaffold control (p = 0.0067). All measurements were done in duplicate on different days and the bars indicate the standard deviation. Analyses of non-treated cells were included in all experiments (background).

Article Snippet: ERBB2-Fc chimera (R&D Systems) was used as target in half of the tracks (C, D, G and H) and biotinylated (using Biotin-XX-succinimidyl ester B-1606, Invitrogen) ERBB2-His 6 (Sino Biological) was used in the remaining tracks (A, B, E and F).

Techniques: Binding Assay, Incubation, Labeling, Standard Deviation, Concentration Assay, Flow Cytometry, Two Tailed Test

Journal: eLife

Article Title: Unique integrated stress response sensors regulate cancer cell susceptibility when Hsp70 activity is compromised

doi: 10.7554/eLife.64977

Figure Lengend Snippet: ( A ) HER2 (diamonds), TNBC (circles), and luminal (squares and triangles) breast cancer lines were seeded into 96-well plates and treated with increasing doses of MAL3-101 for 72 hr. Viability is expressed as the average of three or more independent experiments, ± SEM. ( B ) MAL3-101-sensitive (MCF7 and MDA MB 231, denoted in blue) and resistant (MDA MB 453 and MDA MB 361, denoted in black) cells were treated with 12 µM MAL3-101 for the indicated times, and lysates were prepared and immunoblotted for cleaved caspase-3, caspase-7, and caspase-8. β-actin serves as a loading control. ( C ) The corresponding fold-increase of the indicated apoptotic markers relative to the DMSO control are plotted, ± SEM (n≥3 for cleaved caspase-3, n=3 for cleaved caspase-7, and n≥4 for cleaved caspase-8). Black asterisks correspond to statistical significance between MDA MB 231 cells (closed circle) and MDA MB 453 and MDA MB 361 (open circle and triangle, respectively), and the red asterisk represents statistical significance between MCF7 (closed triangle) and MDA MB 453 and MDA MB 361 (open circle and triangle) cells; * denotes p<0.05, ** denotes p<0.005. Figure 1—source data 1. Source data for cell viability assay and apoptotic marker accumulation in .

Article Snippet: For the remaining proteins aliquots from the same lysates were instead heated to 37°C for 30 min prior to SDS-PAGE using 4–20% Tris-Glycine gradient gels (XP04202Box, Thermo Fisher Scientific), and after transfer on nitrocellulose membranes (#84–874, Prometheus Laboratories Inc, California), the blots were incubated with anti-cleaved Caspase-8 (18C8, #9496; at 1:1000), anti-BiP (C50B12, #3177S; at 1:1000), anti-Hsp70 (smc-113, StressMarq Biosciences, Victoria, British Columbia; at 1:1000), anti-Hsc/Hsp70 (#4872S; at 1:1000), anti-HER2 (29D8, #2165; at 1:2000), anti-eIF2α (#9722S; at 1:2000), anti-phospho-eIF2α (#9721L; at 1:500), anti-PERK (D11A8, #5683S; at 1:2000), anti-PKR (D7F7, #12297S; at 1:2000), anti-Ire1α (14C10, #3294S; at 1:1000), anti-HRI (MBS2538114, MyBioSource, San Diego, CA; at 1:500) and anti-GCN2 and anti-GCN2 pT899 antibody (ab-134053 and ab-75836, Abcam, Cambtidge, UK; at 1:2000).

Techniques: Viability Assay, Marker

Journal: eLife

Article Title: Unique integrated stress response sensors regulate cancer cell susceptibility when Hsp70 activity is compromised

doi: 10.7554/eLife.64977

Figure Lengend Snippet: Breast cancer cells exhibit a range of sensitivities to MAL3-101, a specific Hsp70 inhibitor. The indicated breast cancer lines were seeded into 96-well plates and treated with increasing doses of the indicated compounds for 72 hr. Viability was measured using the CellTiter-Glo assay. IC 50 values were generate using a sigmoidal nonlinear regression with SigmaPlot 11.0. ND stands for an undetermined value. MAL3-101 sensitivities of Hsp70 inhibitor resistant cells are in bold.

Article Snippet: For the remaining proteins aliquots from the same lysates were instead heated to 37°C for 30 min prior to SDS-PAGE using 4–20% Tris-Glycine gradient gels (XP04202Box, Thermo Fisher Scientific), and after transfer on nitrocellulose membranes (#84–874, Prometheus Laboratories Inc, California), the blots were incubated with anti-cleaved Caspase-8 (18C8, #9496; at 1:1000), anti-BiP (C50B12, #3177S; at 1:1000), anti-Hsp70 (smc-113, StressMarq Biosciences, Victoria, British Columbia; at 1:1000), anti-Hsc/Hsp70 (#4872S; at 1:1000), anti-HER2 (29D8, #2165; at 1:2000), anti-eIF2α (#9722S; at 1:2000), anti-phospho-eIF2α (#9721L; at 1:500), anti-PERK (D11A8, #5683S; at 1:2000), anti-PKR (D7F7, #12297S; at 1:2000), anti-Ire1α (14C10, #3294S; at 1:1000), anti-HRI (MBS2538114, MyBioSource, San Diego, CA; at 1:500) and anti-GCN2 and anti-GCN2 pT899 antibody (ab-134053 and ab-75836, Abcam, Cambtidge, UK; at 1:2000).

Techniques:

Journal: eLife

Article Title: Unique integrated stress response sensors regulate cancer cell susceptibility when Hsp70 activity is compromised

doi: 10.7554/eLife.64977

Figure Lengend Snippet: The cell numbers and autophagy or proteasome inhibitor concentrations used for the cell viability assay in combination with increasing doses of MAL3-101 are shown. The concentrations of bortezomib, CQ, and bafilomycin to induce no greater than 30% of cell death in each line after 72 hr treatment are shown. ND stands for undetermined value.

Article Snippet: For the remaining proteins aliquots from the same lysates were instead heated to 37°C for 30 min prior to SDS-PAGE using 4–20% Tris-Glycine gradient gels (XP04202Box, Thermo Fisher Scientific), and after transfer on nitrocellulose membranes (#84–874, Prometheus Laboratories Inc, California), the blots were incubated with anti-cleaved Caspase-8 (18C8, #9496; at 1:1000), anti-BiP (C50B12, #3177S; at 1:1000), anti-Hsp70 (smc-113, StressMarq Biosciences, Victoria, British Columbia; at 1:1000), anti-Hsc/Hsp70 (#4872S; at 1:1000), anti-HER2 (29D8, #2165; at 1:2000), anti-eIF2α (#9722S; at 1:2000), anti-phospho-eIF2α (#9721L; at 1:500), anti-PERK (D11A8, #5683S; at 1:2000), anti-PKR (D7F7, #12297S; at 1:2000), anti-Ire1α (14C10, #3294S; at 1:1000), anti-HRI (MBS2538114, MyBioSource, San Diego, CA; at 1:500) and anti-GCN2 and anti-GCN2 pT899 antibody (ab-134053 and ab-75836, Abcam, Cambtidge, UK; at 1:2000).

Techniques: Viability Assay

Journal: eLife

Article Title: Unique integrated stress response sensors regulate cancer cell susceptibility when Hsp70 activity is compromised

doi: 10.7554/eLife.64977

Figure Lengend Snippet: Breast cancer cells exhibit a range of sensitivities to MAL3-101 in the presence of either autophagy or proteasome inhibitors. Cells were seeded into 96-well plates and treated with increasing doses of MAL3-101 in the presence or absence of subcritical doses of bortezomib (proteasome inhibitor), or CQ or bafilomycin (autophagy inhibitors) for 72 hr. Viability was measured using the CellTiter-Glo assay. IC 50 values were generate using a sigmoidal nonlinear regression with SigmaPlot 11.0. ND stands for undetermined value. MAL3-101 resistant cells are highlighted in yellow.

Article Snippet: For the remaining proteins aliquots from the same lysates were instead heated to 37°C for 30 min prior to SDS-PAGE using 4–20% Tris-Glycine gradient gels (XP04202Box, Thermo Fisher Scientific), and after transfer on nitrocellulose membranes (#84–874, Prometheus Laboratories Inc, California), the blots were incubated with anti-cleaved Caspase-8 (18C8, #9496; at 1:1000), anti-BiP (C50B12, #3177S; at 1:1000), anti-Hsp70 (smc-113, StressMarq Biosciences, Victoria, British Columbia; at 1:1000), anti-Hsc/Hsp70 (#4872S; at 1:1000), anti-HER2 (29D8, #2165; at 1:2000), anti-eIF2α (#9722S; at 1:2000), anti-phospho-eIF2α (#9721L; at 1:500), anti-PERK (D11A8, #5683S; at 1:2000), anti-PKR (D7F7, #12297S; at 1:2000), anti-Ire1α (14C10, #3294S; at 1:1000), anti-HRI (MBS2538114, MyBioSource, San Diego, CA; at 1:500) and anti-GCN2 and anti-GCN2 pT899 antibody (ab-134053 and ab-75836, Abcam, Cambtidge, UK; at 1:2000).

Techniques:

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: TWEAK/Fn14 disrupts Th17/Treg balance and aggravates conjunctivitis by inhibiting the Nrf2/HO-1 pathway in allergic conjunctivitis mice.

doi: 10.1186/s10020-024-01004-5

Figure Lengend Snippet: Fig. 3 TWEAK regulated the Th17/Treg cell ratio in AC mice. (A) The effect of TWEAK knockdown on Th17 and Treg cell ratio in spleen of AC mice was observed by flow cytometry. (B-C) WB and IHC were employed to evaluate the expression levels of FoxP3 and RORγt in mice conjunctival tissue. (D) The effect of TWEAK knockdown on the levels of Th17 and Treg cytokines in mice spleen were evaluated by ELISA. *p < 0.05, **p < 0.01, ***p < 0.001 vs. Con + AAV-shNC; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. AC + AAV-shNC; ns, no significant difference

Article Snippet: After deparaffinization and rehydration, tissue slides were treated with 100 μL endogenous peroxidase blockers for 10 min. Then, the slides were incubated with primary antibody against RORγt (14-6981-82, 1:200; ThermoFisher, Waltham, MA, USA), FoxP3 (BA2032-1, 1:200; Boster, Pleasanton, CA, USA), TWEAK (BM4635, 1:200; Boster), Fn14 (GTX85216; 1:200, Genetex, Alton, CA, USA), Nrf2 (PA5-27882; 1:200, ThermoFisher) and HO-1 (PA5-77833, 1:200; ThermoFisher) overnight at 4 °C.

Techniques: Knockdown, Flow Cytometry, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: TWEAK/Fn14 disrupts Th17/Treg balance and aggravates conjunctivitis by inhibiting the Nrf2/HO-1 pathway in allergic conjunctivitis mice.

doi: 10.1186/s10020-024-01004-5

Figure Lengend Snippet: Fig. 6 Inhibition of Nrf2/HO-1 signaling pathway affected Th17/Treg cell ratio in AC mice with TWEAK knockdown. (A) The effect of Nrf2 inhibitor on Th17/Treg cell ratio in AC mice with TWEAK knockdown was assessed by flow cytometry. (B-C) WB and IHC assays were employed to evaluate the expres sion of FoxP3 and RORγt in conjunctival tissue of AC mice. (D) The levels of Th17 and Treg cytokines in mice spleen were detected by ELISA. *p < 0.05, **p < 0.01, ***p < 0.001 vs. AC + AAV-shNC; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. AC + AAV-shTWEAK

Article Snippet: After deparaffinization and rehydration, tissue slides were treated with 100 μL endogenous peroxidase blockers for 10 min. Then, the slides were incubated with primary antibody against RORγt (14-6981-82, 1:200; ThermoFisher, Waltham, MA, USA), FoxP3 (BA2032-1, 1:200; Boster, Pleasanton, CA, USA), TWEAK (BM4635, 1:200; Boster), Fn14 (GTX85216; 1:200, Genetex, Alton, CA, USA), Nrf2 (PA5-27882; 1:200, ThermoFisher) and HO-1 (PA5-77833, 1:200; ThermoFisher) overnight at 4 °C.

Techniques: Inhibition, Knockdown, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: The unique monoclonal antibodies and immunochemical assay for comprehensive determination of the cell-bound and soluble HER2 in different biological samples

doi: 10.1038/s41598-024-54590-z

Figure Lengend Snippet: Unique fragments of the CDRs within the heavy and light chains of the new mouse monoclonal anti-HER2 antibody. The amino acid sequence corresponds to the characteristic nucleotide sequence of (A ) anti-human HER2/70.27.58 mAb and (B ) anti-human HER2/70.21.73.67 mAb.

Article Snippet: Additional tests were conducted on plates coated with recombinant HER2 His-tag (ProSci, Fort Collins, CO, USA) to ensure antibody specificity.

Techniques: Sequencing

Journal: Scientific Reports

Article Title: The unique monoclonal antibodies and immunochemical assay for comprehensive determination of the cell-bound and soluble HER2 in different biological samples

doi: 10.1038/s41598-024-54590-z

Figure Lengend Snippet: Anti-HER2 monoclonal antibodies production and characterization. ( A ) Morphology of the anti-human HER2/70.27.58 and anti-human HER2/70.21.73.67 hybridoma cells photographed at 20 × and 40 × magnification. ( B ) FPLC chromatograms were recorded during the purification of the anti-human HER2/70.27.58 and anti-human HER2/70.21.73.67 antibodies using affinity chromatography on the Protein A resin. ( C ) SDS-PAGE analysis of the purified anti-human HER2/70.27.58 and anti-human HER2/70.21.73.67 antibodies loaded at the amount of 1 µg/well on the 12% polyacrylamide gel under reducing conditions. ( D ) WB analysis of HER2 in whole cell lysates of the HER2 low expressing (MDA-MB-231) and HER2 high expressing (SK-BR-3, SK-OV-3) cells, probed with the home-made anti-human HER2/70.27.58 monoclonal antibody and detected with the secondary anti-mouse IgG-HRP (upper panel). The recombinant HER2 ECD protein was used as a reference. The loading control was performed with membrane probed with antibody binding \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\upbeta$$\end{document} β -actin (lower panel). ( E ) The formaldehyde-fixed SK-OV-3 cells were photographed in the bright field (BF) at the 40 × magnification. Immunofluorescence analysis was performed on cells stained with the commercial anti-HER2 ECD antibody followed by anti-mouse IgG-AlexaFluor594 (AF594) (red channel) and co-stained with the anti-HER2/70.27.58 or anti-HER2/70.21.73.67 antibodies detected with the AlexaFluor488-labeled (AF488) secondary antibody (green channel). Nuclei were stained with DAPI (blue channel) ( F ) Quantitative ELISA with the anti-human HER2/70.27.58 and anti-human HER2/70.21.73.67 antibodies loaded in a range of 0–5 µg/ml on the plate coated with the recombinant chimera of the HER2 ECD-Fc protein. The signal generated from secondary antibody anti-mouse IgG-HRP was quantified by measuring absorbance at 450 nm and expressed after background subtraction (A 450 -A 0 ).

Article Snippet: Additional tests were conducted on plates coated with recombinant HER2 His-tag (ProSci, Fort Collins, CO, USA) to ensure antibody specificity.

Techniques: Bioprocessing, Purification, Affinity Chromatography, SDS Page, Expressing, Recombinant, Control, Membrane, Binding Assay, Immunofluorescence, Staining, Labeling, Enzyme-linked Immunosorbent Assay, Generated

Journal: Scientific Reports

Article Title: The unique monoclonal antibodies and immunochemical assay for comprehensive determination of the cell-bound and soluble HER2 in different biological samples

doi: 10.1038/s41598-024-54590-z

Figure Lengend Snippet: Parameters of the sandwich ELISA for HER2 detection. ( A ) HER2 binding kinetics in the standard curve concentration range of 0.156–10 000 ng/well (1.56–100 ng/ml). Results are expressed as absorbance at 450 nm after background subtraction (A 450 -A 0 ). ( B ) Assay accuracy was tested by comparison of HER2 level measured by ELISA in the samples of the known antigen concentration (mock samples). Data were collected for 2, 5, 10, 30, and 50 ng/ml of HER2 (given concentration; x-axis ), covering both physiological and increased concentrations. Experimentally measured concentration [ng/ml] is shown on the y-axis . Error bars indicate SD.

Article Snippet: Additional tests were conducted on plates coated with recombinant HER2 His-tag (ProSci, Fort Collins, CO, USA) to ensure antibody specificity.

Techniques: Sandwich ELISA, Binding Assay, Concentration Assay, Comparison, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: The unique monoclonal antibodies and immunochemical assay for comprehensive determination of the cell-bound and soluble HER2 in different biological samples

doi: 10.1038/s41598-024-54590-z

Figure Lengend Snippet: HER2 levels in the cell culture medium and whole cell lysates of MDA-MB-231, SK-OV-3, and SK-BR-3 cells measured by in-house ELISA.

Article Snippet: Additional tests were conducted on plates coated with recombinant HER2 His-tag (ProSci, Fort Collins, CO, USA) to ensure antibody specificity.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: The unique monoclonal antibodies and immunochemical assay for comprehensive determination of the cell-bound and soluble HER2 in different biological samples

doi: 10.1038/s41598-024-54590-z

Figure Lengend Snippet: HER2 expression in tumors from mice with xenografted human cancer cells. ( A ) Immunohistochemistry staining using anti-HER2/70.27.58 mAb of the mouse tumors induced with the human ovarian cancer cells (SK-OV-3) overexpressing HER2 and ( B ) human epithelial breast cancer cells (MDA-MB-231) with low expression of HER.

Article Snippet: Additional tests were conducted on plates coated with recombinant HER2 His-tag (ProSci, Fort Collins, CO, USA) to ensure antibody specificity.

Techniques: Expressing, Immunohistochemistry, Staining

Journal: Scientific Reports

Article Title: The unique monoclonal antibodies and immunochemical assay for comprehensive determination of the cell-bound and soluble HER2 in different biological samples

doi: 10.1038/s41598-024-54590-z

Figure Lengend Snippet: HER2 levels in the mice sera and tumors from mice xenografted with human cancer cells.

Article Snippet: Additional tests were conducted on plates coated with recombinant HER2 His-tag (ProSci, Fort Collins, CO, USA) to ensure antibody specificity.

Techniques:

Journal: Scientific Reports

Article Title: The unique monoclonal antibodies and immunochemical assay for comprehensive determination of the cell-bound and soluble HER2 in different biological samples

doi: 10.1038/s41598-024-54590-z

Figure Lengend Snippet: HER2 measurements in the human serum samples from patients and control donors.

Article Snippet: Additional tests were conducted on plates coated with recombinant HER2 His-tag (ProSci, Fort Collins, CO, USA) to ensure antibody specificity.

Techniques: Control, Enzyme-linked Immunosorbent Assay