Journal: Cell Death & Disease

Article Title: UBIAD1 suppresses the proliferation of bladder carcinoma cells by regulating H-Ras intracellular trafficking via interaction with the C-terminal domain of H-Ras

doi: 10.1038/s41419-018-1215-4

Figure Lengend Snippet: a UBIAD1 reduced cell viability in T24 bladder cancer cells. T24 cells were transiently transfected with pcDNA3.1-UBIAD1 plasmid. Twenty-four hours after transfection, cell viability was detected by the MTT assay. *** p < 0.001, Student’s t -test, n = 6 experiments. b UBIAD1 inhibited the proliferation of T24 cells. T24 cells were transiently transfected with pcDNA3.1-UBIAD1 plasmid. Twenty-four hours after transfection, cell proliferation was detected using the BrdU cell proliferation ELISA kit. ** p < 0.01, Student’s t -test, n = 3 experiments. c UBIAD1 inhibited the growth of T24 cells. T24 cells were transiently transfected with pcDNA3.1-UBIAD1 plasmid. Twenty-four hours after transfection, cell growth was detected by cell counting. ** p < 0.01, Student’s t -test, n = 3 experiments. d UBIAD1 inhibits ERK phosphorylation in T24 cells. T24 cells were transfected with increasing amounts of pcDNA3.1-UBIAD1. Twenty-four hours after transfection, total cell lysate was examined by western blotting (WB). The same experiment was repeated at least three times and representative data are shown. This protocol is the same for most immunoblotting analyses throughout the study. The right panel represents the ratio (mean ± SD) from densitometry analyses. ** p < 0.01, *** p < 0.001, Student’s t -test. e The Ras/ERK signaling pathway is activated by UBIAD1 deficiency in HEK293T cells. HEK293T cells were transfected with sh-UBIAD1. Seventy-two hours after transfection, total cell lysate was exposed to antibodies and WB was performed, as indicated. The same experiment was repeated three times. f Upregulated p-ERK was abrogated by GFP-RBD. HEK293T cells were transfected with plasmids as indicated. After 72 h of transfection, the total cell lysate was exposed to antibodies and examined by WB. The same experiment was repeated three times. g UBIAD1 inhibited H-Ras-induced p-ERK. HEK293T cells were transfected with plasmids as indicated. Twenty-four hours, the total cell lysate was first exposed to antibodies and then examined by WB. The same experiment was repeated three times. The right panel represents the ratio (mean ± SD) from densitometer analyses. ** p < 0.01, Student’s t -test. h The mutation of heix ( heix k11403 /Df and heix k11403 /heix 1 ) produced melanotic masses in Drosophila larvae; w 1118 is the wild-type and heix k11403 /Df; tub P > heix is the rescue type. Melanotic masses was detected in long larvae following crosses performed for 2 weeks. N.D.: not detected, ( n = 3 experiments, each with 50 larvae). i The mutation of heix increased p-ERK in Drosophila larvae. Total larvae lysate was exposed to antibodies and examined by WB as indicated in the material and methods. The same experiment was repeated three times. j Melanotic masses disappeared under U0126 treatment in the heix mutant larvae. Melanotic masses were detected in long larvae following 2 weeks crosses. *** p < 0.001, Student’s t -test, ( n = 3 experiments, each with 50 larvae)

Article Snippet: The following antibodies were used in this study: p-c-Raf (Ser259) (#9421), p-c-Raf (Ser338) (#9427), p-c-Raf (Ser289/296/301) (#9431), c-Raf (#9422), p-ERK1/2 (#4370), total-ERK1/2 (#4695), and caspase-3 (#9662) from Cell Signaling Technology (USA); anti-phospho-MEK (Ser218/222)/MEK2 (Ser222/226) (#2283379) from Millipore (USA); MEK1/2 (Ab-217/221) (#21203) antibody from Signalway Antibody (USA); Antibodies detecting actin (A01010-1) and GAPDH (A01020-1) from Abbkine (USA); Flag tag monoclonal antibody (A00187) from GenScript (China); GFP tag antibody (#66002-1) and HMGB1 (#10829-1) from proteintech (China); 488/549/645-conjugated secondary antibody from Abbkine (USA); HRP-conjugated secondary antibody from ABGENT (USA); UBIAD1 antibody (ab36832), Pan-Ras antibody (ab16907), and LC3B (AB192890) from Abcam (USA); rabbit IgG, mouse IgG from Immunoreagents (USA).

Techniques: Transfection, Plasmid Preparation, MTT Assay, Enzyme-linked Immunosorbent Assay, Cell Counting, Phospho-proteomics, Western Blot, Mutagenesis, Produced

Journal: Cell Death & Disease

Article Title: UBIAD1 suppresses the proliferation of bladder carcinoma cells by regulating H-Ras intracellular trafficking via interaction with the C-terminal domain of H-Ras

doi: 10.1038/s41419-018-1215-4

Figure Lengend Snippet: a UBIAD1 increased H-Ras retention in the Golgi apparatus in HEK293T cells. HEK293T cells were transfected with RFP-H-Ras with or without UBIAD1-GFP, followed by 24 h of culturing with or without cycloheximide for 5 h and confocal analysis. Histograms show the percentage of H-Ras localized in the Golgi. ** p < 0.01 compared to control, ( n = 3 experiments, each with 100 cells). b Ectopic expression of UBIAD1 increased the localization of Ras in the Golgi apparatus. T24 cells were transfected with or without UBIAD1-GFP, followed by 24 h of culture, stained with pan-Ras antibody and analyzed by confocal microscopy. Histograms show the percentage of Ras localized in the Golgi. ** p < 0.01 compared to control, ( n = 3 experiments, each with 100 cells). c Ras localized in the plasma membrane after knockdown of UBIAD1. HEK293T cells were transfected with sh-UBIAD1, followed by 72 h of culturing, staining with DiI (a maker of membrane), pan-Ras antibody and DAPI and confocal analysis. Histograms show the percentage of Ras localized in the plasma membrane. ** p < 0.01 compared to control, ( n = 3 experiments, each with 100 cells)

Article Snippet: The following antibodies were used in this study: p-c-Raf (Ser259) (#9421), p-c-Raf (Ser338) (#9427), p-c-Raf (Ser289/296/301) (#9431), c-Raf (#9422), p-ERK1/2 (#4370), total-ERK1/2 (#4695), and caspase-3 (#9662) from Cell Signaling Technology (USA); anti-phospho-MEK (Ser218/222)/MEK2 (Ser222/226) (#2283379) from Millipore (USA); MEK1/2 (Ab-217/221) (#21203) antibody from Signalway Antibody (USA); Antibodies detecting actin (A01010-1) and GAPDH (A01020-1) from Abbkine (USA); Flag tag monoclonal antibody (A00187) from GenScript (China); GFP tag antibody (#66002-1) and HMGB1 (#10829-1) from proteintech (China); 488/549/645-conjugated secondary antibody from Abbkine (USA); HRP-conjugated secondary antibody from ABGENT (USA); UBIAD1 antibody (ab36832), Pan-Ras antibody (ab16907), and LC3B (AB192890) from Abcam (USA); rabbit IgG, mouse IgG from Immunoreagents (USA).

Techniques: Transfection, Control, Expressing, Staining, Confocal Microscopy, Clinical Proteomics, Membrane, Knockdown

Journal: Cell Death & Disease

Article Title: UBIAD1 suppresses the proliferation of bladder carcinoma cells by regulating H-Ras intracellular trafficking via interaction with the C-terminal domain of H-Ras

doi: 10.1038/s41419-018-1215-4

Figure Lengend Snippet: a UBIAD1 changed the localization of H-Ras under short-term (5 min) EGF treatment. HEK293T cells were transfected with plasmids as indicated. After 24 h of transfection, the cells were serum-starved for 6 h before treatment with EGF (100 ng/ml) for different periods of time, followed by confocal analysis, the same experiment was repeated three times. b UBIAD1 decreased short-term EGF-induced p-ERK. HEK293T cells were transfected with plasmids as indicated. Twenty-four hours after transfection, the cells were serum-starved for 6 h before treatment with EGF (100 ng/ml) for different periods of time. The total cell lysate was exposed to antibodies and examined by WB as indicated. The same experiment was repeated three times. c Ras aggregates in the plasma membrane after knocking down UBIAD1. HEK293T cells were transfected with sh-UBIAD1, followed by 72 h of culture and 24 h of culturing with or without 2BP, staining with pan-Ras antibody and confocal analysis. The same experiment was repeated three times. d Ras inhibitors reduced ERK phosphorylation after UBIAD1 knockdown. HEK293T cells were transfected with sh-UBIAD1, followed by 72 h of culture with or without Ras inhibitors (FTI-277, Salirasib, 2BP, tunicamycin). The total cell lysate was exposed to antibodies and WB was performed as indicated. The same experiment was repeated three times. e Melanotic masses disappeared under Ras inhibitor treatment in the heix mutant larvae. Melanotic masses were detected in long larvae following 2 weeks crosses. * p < 0.05, *** p < 0.001, Student’s t -test, ( n = 3 experiments, each with 50 larvae)

Article Snippet: The following antibodies were used in this study: p-c-Raf (Ser259) (#9421), p-c-Raf (Ser338) (#9427), p-c-Raf (Ser289/296/301) (#9431), c-Raf (#9422), p-ERK1/2 (#4370), total-ERK1/2 (#4695), and caspase-3 (#9662) from Cell Signaling Technology (USA); anti-phospho-MEK (Ser218/222)/MEK2 (Ser222/226) (#2283379) from Millipore (USA); MEK1/2 (Ab-217/221) (#21203) antibody from Signalway Antibody (USA); Antibodies detecting actin (A01010-1) and GAPDH (A01020-1) from Abbkine (USA); Flag tag monoclonal antibody (A00187) from GenScript (China); GFP tag antibody (#66002-1) and HMGB1 (#10829-1) from proteintech (China); 488/549/645-conjugated secondary antibody from Abbkine (USA); HRP-conjugated secondary antibody from ABGENT (USA); UBIAD1 antibody (ab36832), Pan-Ras antibody (ab16907), and LC3B (AB192890) from Abcam (USA); rabbit IgG, mouse IgG from Immunoreagents (USA).

Techniques: Transfection, Clinical Proteomics, Membrane, Staining, Phospho-proteomics, Knockdown, Mutagenesis

Journal: Cell Death & Disease

Article Title: UBIAD1 suppresses the proliferation of bladder carcinoma cells by regulating H-Ras intracellular trafficking via interaction with the C-terminal domain of H-Ras

doi: 10.1038/s41419-018-1215-4

Figure Lengend Snippet: a Ectopically expressed UBIAD1 colocalized with H-Ras in the Golgi apparatus. HEK293T cells were transfected with UBIAD1-GFP, Golgi-RFP and BFP-H-Ras, followed by 24 h of cell culture and confocal analysis. The same experiment was repeated three times. b Fluorescence resonance energy transfer (FRET) between UBIAD1 and H-Ras. HEK293T cells were transfected with plasmids as indicated, followed by 24 h of culture, confocal microscopy and PixFRET analysis by ImageJ software. The arrows indicate the FRET signal. Positive control: pCasper-BG (a standard probe showing the BFP-GFP FRET phenomenon). Negative control: BFP and GFP. *** p < 0.001, Student’s t -test, ( n = 3 experiments, each with 50 cells). c Interaction of UBIAD1 with H-Ras by co-IP. HEK293T cells were transiently transfected with plasmids as indicated. Forty-eight hours after transfection, the cell lysate was exposed to antibodies and analyzed by IP and IB as indicated. The same experiment was repeated three times. d Interaction of endogenous UBIAD1 with pan-Ras by co-IP. The total cell lysate from HEK293T cells was exposed to rabbit anti-UBIAD1 antibody and analyzed by IP, followed by IB. Rabbit IgG was used as a negative control. The same experiment was repeated three times. e Interaction of endogenous HEIX with pan-Ras in wild-type Drosophila larvae by co-IP. The total cell lysate from larvae was exposed to rabbit anti-HEIX antibody or rabbit IgG and analyzed by IP followed by IB as indicated. The same experiment was repeated three times

Article Snippet: The following antibodies were used in this study: p-c-Raf (Ser259) (#9421), p-c-Raf (Ser338) (#9427), p-c-Raf (Ser289/296/301) (#9431), c-Raf (#9422), p-ERK1/2 (#4370), total-ERK1/2 (#4695), and caspase-3 (#9662) from Cell Signaling Technology (USA); anti-phospho-MEK (Ser218/222)/MEK2 (Ser222/226) (#2283379) from Millipore (USA); MEK1/2 (Ab-217/221) (#21203) antibody from Signalway Antibody (USA); Antibodies detecting actin (A01010-1) and GAPDH (A01020-1) from Abbkine (USA); Flag tag monoclonal antibody (A00187) from GenScript (China); GFP tag antibody (#66002-1) and HMGB1 (#10829-1) from proteintech (China); 488/549/645-conjugated secondary antibody from Abbkine (USA); HRP-conjugated secondary antibody from ABGENT (USA); UBIAD1 antibody (ab36832), Pan-Ras antibody (ab16907), and LC3B (AB192890) from Abcam (USA); rabbit IgG, mouse IgG from Immunoreagents (USA).

Techniques: Transfection, Cell Culture, Fluorescence, Förster Resonance Energy Transfer, Confocal Microscopy, Software, Positive Control, Negative Control, Co-Immunoprecipitation Assay

Journal: Cell Death & Disease

Article Title: UBIAD1 suppresses the proliferation of bladder carcinoma cells by regulating H-Ras intracellular trafficking via interaction with the C-terminal domain of H-Ras

doi: 10.1038/s41419-018-1215-4

Figure Lengend Snippet: a FNTA knockdown decreased interaction between UBIAD1 and H-Ras. HEK293T cells were transfected with 3Flag-UBIAD1 and GFP-H-Ras with or without sh-FNTA. Seventy-two hours after transfection, the cell lysate was exposed to antibodies and analyzed by IP and IB as indicated. The same experiment was repeated three times. b Icmt knockdown decreased interaction between UBIAD1 and H-Ras. HEK293T cells were transfected with 3Flag-UBIAD1 and GFP-H-Ras with or without si-Icmt. Seventy-two hours after transfection, the cell lysate was exposed to antibodies and analyzed by IP and IB as indicated. The same experiment was repeated three times. c 2-BP treatment increased interaction between UBIAD1 and H-Ras. HEK293T cells were transfected with 3Flag-UBIAD1 and GFP-H-Ras, followed by 72 h of culture and 24 h of culturing with or without 2BP. The cell lysate was exposed to antibodies and analyzed by IP and IB as indicated. The same experiment was repeated three times. d Interaction of UBIAD1 with truncated H-Ras. HEK293T cells were transiently transfected with plasmids as indicated. Forty-eight hours after transfection, the cell lysate was exposed to antibodies and analyzed by IP and IB as indicated. The same experiment was repeated three times. e The C-terminus of H-Ras decreased interaction between UBIAD1 and H-Ras. HEK293T cells were transiently transfected with plasmids as indicated. Forty-eight hours after transfection, the cell lysate was exposed to antibodies and analyzed by IP and IB as indicated. The same experiment was repeated three times. f The C-terminus of H-Ras blocked UBIAD1 function with decreases in p-ERK levels. HEK293T or T24 cells were transfected with plasmids as indicated. Twenty-four hours after transfection, the total cell lysate was analyzed by WB. The lower panel presents the ratio (mean ± SD) from densitometry analyses. ** p < 0.01, ns: not significant, Student’s t -test, n = 3 experiments. g The C-terminus of H-Ras suppressed the UBIAD1-induced decrease in T24 cell viability. T24 cells were transfected with plasmids as indicated. Cell viability was detected by the MTT assay. ** p < 0.01, Student’s t -test, n = 3 experiments. h The C-terminus of H-Ras inhibited the UBIAD1-induced decrease in T24 cell proliferation. T24 cells were transfected with the plasmids as indicated. Cell proliferation was detected using the BrdU cell proliferation ELISA kit. ** p < 0.01, Student’s t -test, n = 3 experiments. i The C-terminus of H-Ras inhibited the UBIAD1-induced decrease in T24 cell growth. T24 cells were transfected with plasmids as indicated. Cell growth was detected by cell counting, n = 3 experiments

Article Snippet: The following antibodies were used in this study: p-c-Raf (Ser259) (#9421), p-c-Raf (Ser338) (#9427), p-c-Raf (Ser289/296/301) (#9431), c-Raf (#9422), p-ERK1/2 (#4370), total-ERK1/2 (#4695), and caspase-3 (#9662) from Cell Signaling Technology (USA); anti-phospho-MEK (Ser218/222)/MEK2 (Ser222/226) (#2283379) from Millipore (USA); MEK1/2 (Ab-217/221) (#21203) antibody from Signalway Antibody (USA); Antibodies detecting actin (A01010-1) and GAPDH (A01020-1) from Abbkine (USA); Flag tag monoclonal antibody (A00187) from GenScript (China); GFP tag antibody (#66002-1) and HMGB1 (#10829-1) from proteintech (China); 488/549/645-conjugated secondary antibody from Abbkine (USA); HRP-conjugated secondary antibody from ABGENT (USA); UBIAD1 antibody (ab36832), Pan-Ras antibody (ab16907), and LC3B (AB192890) from Abcam (USA); rabbit IgG, mouse IgG from Immunoreagents (USA).

Techniques: Knockdown, Transfection, MTT Assay, Enzyme-linked Immunosorbent Assay, Cell Counting

Journal: Cell Death & Disease

Article Title: UBIAD1 suppresses the proliferation of bladder carcinoma cells by regulating H-Ras intracellular trafficking via interaction with the C-terminal domain of H-Ras

doi: 10.1038/s41419-018-1215-4

Figure Lengend Snippet: a GGPP supplementation prevented a reduction in interaction between UBIAD1 and H-Ras in the absence of GGPPS. HEK293T cells were transfected with 3Flag-UBIAD1, GFP-H-Ras, and si-GGPPS as indicated, followed by 72 h of culturing with or without GGPP, and all lysates were analyzed by IP and IB. The same experiment was repeated three times. b GGPPS knockdown abolished UBIAD1-induced pan-Ras retention in the Golgi. T24 cells were transfected with si-GGPPS and after 48 h, the same cells were transfected with the UBIAD1-GFP construct. The cells were cultured with or without GGPP, followed by staining for pan-Ras and confocal analysis. The same experiment was repeated three times. c GGPPS knockdown abolished the UBIAD1-induced decrease in ERK phosphorylation. HEK293T or T24 cells were transfected with si-GGPPS and after 48 h, the same cells were transfected with plasmids as indicated. The cells were cultured with or without GGPP. The total cell lysate was analyzed by WB. The lower panel presents the ratio (mean ± SD) from densitometry analyses. ** p < 0.01, Student’s t -test, n = 3 experiments. d GGPPS knockdown suppressed the UBIAD1-induced decrease in T24 cell viability. T24 cells were transfected with si-GGPPS and after 48 h, the same cells were transfected with pcDNA3.1-UBIAD1. The cells were cultured with or without GGPP. Cell viability was detected by the MTT assay. ** p < 0.01, *** p < 0.001, Student’s t -test, n = 3 experiments. e GGPPS knockdown suppresses the UBIAD1-induced decrease in T24 cell viability. T24 cells were transfected with si-GGPPS and after 48 h, the same cells were transfected with pcDNA3.1-UBIAD1. The cells were cultured with or without GGPP. Cell proliferation was detected using the BrdU cell proliferation ELISA kit. ** p < 0.01, Student’s t -test, n = 3 experiments. f GGPPS knockdown suppresses the UBIAD1-induced decrease in T24 cell growth. T24 cells were transfected with si-GGPPS and after 48 h, the same cells were transfected with pcDNA3.1-UBIAD1. The cells were cultured with or without GGPP. Cell growth was detected by cell counting, n = 3 experiments

Article Snippet: The following antibodies were used in this study: p-c-Raf (Ser259) (#9421), p-c-Raf (Ser338) (#9427), p-c-Raf (Ser289/296/301) (#9431), c-Raf (#9422), p-ERK1/2 (#4370), total-ERK1/2 (#4695), and caspase-3 (#9662) from Cell Signaling Technology (USA); anti-phospho-MEK (Ser218/222)/MEK2 (Ser222/226) (#2283379) from Millipore (USA); MEK1/2 (Ab-217/221) (#21203) antibody from Signalway Antibody (USA); Antibodies detecting actin (A01010-1) and GAPDH (A01020-1) from Abbkine (USA); Flag tag monoclonal antibody (A00187) from GenScript (China); GFP tag antibody (#66002-1) and HMGB1 (#10829-1) from proteintech (China); 488/549/645-conjugated secondary antibody from Abbkine (USA); HRP-conjugated secondary antibody from ABGENT (USA); UBIAD1 antibody (ab36832), Pan-Ras antibody (ab16907), and LC3B (AB192890) from Abcam (USA); rabbit IgG, mouse IgG from Immunoreagents (USA).

Techniques: Transfection, Knockdown, Construct, Cell Culture, Staining, Phospho-proteomics, MTT Assay, Enzyme-linked Immunosorbent Assay, Cell Counting

Journal: Cell Death & Disease

Article Title: UBIAD1 suppresses the proliferation of bladder carcinoma cells by regulating H-Ras intracellular trafficking via interaction with the C-terminal domain of H-Ras

doi: 10.1038/s41419-018-1215-4

Figure Lengend Snippet: a A mutation in UBIAD1 (N102S, RPWS > AAAA) could not induce H-Ras retention in the Golgi. UBAD1 N102S cannot use GGPP as a substrate, and UBIAD1 RPWS is not located in the Golgi. HEK293T cells were transfected with plasmids as indicated and after 24 h of transfection, confocal analysis was performed, the same experiment was repeated three times. b A UBIAD1 mutation did not decrease phosphorylated ERK. Cells were transfected with plasmids and 24 h after transfection, total cell lysate was analyzed by WB. The same experiment was repeated three times. c A UBIAD1 mutation did not decrease T24 cell viability. T24 cells were transfected with plasmids and 24 h after transfection, cell viability was detected by the MTT assay. *** p < 0.001, Student’s t -test, n = 3 experiments. d A UBIAD1 mutation did not decrease T24 cell proliferation. T24 cells were transfected with plasmids and 24 h after transfection, cell proliferation was detected by BrdU cell proliferation ELISA kit. *** p < 0.001, Student’s t -test, n = 3 experiments. e A UBIAD1 mutation did not decrease T24 cell growth. T24 cells were transfected with plasmids and 24 h after transfection, cell growth was detected by cell counting, n = 3 experiments

Article Snippet: The following antibodies were used in this study: p-c-Raf (Ser259) (#9421), p-c-Raf (Ser338) (#9427), p-c-Raf (Ser289/296/301) (#9431), c-Raf (#9422), p-ERK1/2 (#4370), total-ERK1/2 (#4695), and caspase-3 (#9662) from Cell Signaling Technology (USA); anti-phospho-MEK (Ser218/222)/MEK2 (Ser222/226) (#2283379) from Millipore (USA); MEK1/2 (Ab-217/221) (#21203) antibody from Signalway Antibody (USA); Antibodies detecting actin (A01010-1) and GAPDH (A01020-1) from Abbkine (USA); Flag tag monoclonal antibody (A00187) from GenScript (China); GFP tag antibody (#66002-1) and HMGB1 (#10829-1) from proteintech (China); 488/549/645-conjugated secondary antibody from Abbkine (USA); HRP-conjugated secondary antibody from ABGENT (USA); UBIAD1 antibody (ab36832), Pan-Ras antibody (ab16907), and LC3B (AB192890) from Abcam (USA); rabbit IgG, mouse IgG from Immunoreagents (USA).

Techniques: Mutagenesis, Transfection, MTT Assay, Enzyme-linked Immunosorbent Assay, Cell Counting

Journal: Nature Biomedical Engineering

Article Title: Antibody-displaying extracellular vesicles for targeted cancer therapy

doi: 10.1038/s41551-024-01214-6

Figure Lengend Snippet: a , Flow cytometry measurement of uptake by MFI of mNG + EVs (Fc-EVs versus ctrl-EVs versus no EVs) in HER2 positive breast cancer cells (SKBR-3 cells) when incubated with HER2-Ab (trastuzumab), control-Ab (IgG ctrl) or no Ab, showing significant increase in uptake of Fc-EVs by trastuzumab. b , Flow cytometry measurement of uptake (by MFI) of mNG + Fc-EVs decorated with the PD-L1-Ab atezolizumab in PD-L1 expression stimulated (IFNγ) or unstimulated malignant melanoma (B16F10) cells. c , Fluorescence microscopy images of B16F10 cells stained with DAPI (blue), untreated (UT) or treated with mNG + (green) Fc-EVs alone or with the PD-L1-Ab atezolizumab, showing increased uptake of the Fc-EVs when decorated with PD-L1-Ab. Panels a and b are shown as mean ± s.d. n = 3 biological replicates. Statistical significance was calculated using one-way ( b ) or two-way ( a ) ANOVA with Tukey’s post-test compared with each value; P values are indicated above the plots throughout.

Article Snippet: The tissues were processed as described in ref. , with lysed tissue and nLuc + EV input being analysed for nanoluc luminescence or for single-cell suspension for flow cytometry, as described above, but with the HER2 Ab (R&D Systems, FAB9896V-100UG) used for flow cytometry applications.

Techniques: Flow Cytometry, Incubation, Control, Expressing, Fluorescence, Microscopy, Staining

Journal: Nature Biomedical Engineering

Article Title: Antibody-displaying extracellular vesicles for targeted cancer therapy

doi: 10.1038/s41551-024-01214-6

Figure Lengend Snippet: IV injection of Fc-EVs with HER2-Ab (trastuzumab, Fc-EV + HER2-Ab) compared to control-Ab (Fc-EV + IgG-ctrl) in HER2 + breast cancer (SKBR-3) tumour-bearing Swiss nude mice. a , The experimental set-up with inoculation of SKBR-3 cells followed by tumour formation for 2 months before IV injection of nLuc + Fc-EVs with Abs, followed by tissue collection 30 min post injection. b , Fold change in detected EVs (based on luminescence) per gram tumour tissue compared to Fc-EV + IgG-ctrl. c , d , Accumulation of Fc-EV + HER2-Ab (based on luminescence) compared to Fc-EV + IgG-ctrl per gram spleen ( c ) and liver ( d ). All data are shown as mean ± s.d. n = 10 mice. Statistical significance was calculated using two-tailed unpaired t -test analysis compared with each value; P values are indicated above the plots throughout.

Article Snippet: The tissues were processed as described in ref. , with lysed tissue and nLuc + EV input being analysed for nanoluc luminescence or for single-cell suspension for flow cytometry, as described above, but with the HER2 Ab (R&D Systems, FAB9896V-100UG) used for flow cytometry applications.

Techniques: IV Injection, Control, Injection, Two Tailed Test

Journal: eNeuro

Article Title: The Progestin Receptor Interactome in the Female Mouse Hypothalamus: Interactions with Synaptic Proteins Are Isoform Specific and Ligand Dependent

doi: 10.1523/ENEURO.0272-17.2017

Figure Lengend Snippet: List of antibodies used for RPPA analysis

Article Snippet: ErbB2/HER2(Y1248)_R_V , Imgenex , IMG-90189 , Rabbit.

Techniques: Plasmid Preparation

Journal: EBioMedicine

Article Title: Antibody-drug conjugate T-DM1 treatment for HER2+ breast cancer induces ROR1 and confers resistance through activation of Hippo transcriptional coactivator YAP1

doi: 10.1016/j.ebiom.2019.04.061

Figure Lengend Snippet: Overexpression of ROR1/YAP1 in breast cancer is associated with poor overall survival outcomes. (a) Graphical representation of cancer types in which ROR1 is most frequently altered in the TCGA dataset. (b) ROR1 and YAP1 mutation analysis of 960 breast cancer samples in the TCGA dataset. (c) Significant co-occurrences of ROR1 and YAP1 in the TCGA dataset. (d) Boxplot comparing ROR1 and YAP1 expression in ER + PR+/HER2-; ER + PR+/HER2+; ER-PR-/HER2+; subtypes samples in the TCGA dataset (one-way ANOVA). (e) Immunohistochemical detection of ROR1 and YAP1 in disease free, ER + PR+/HER2-; ER + PR+/HER2+; ER-PR-/HER2+; breast cancer subtypes from our own data cohort (one-way ANOVA). The boxes show the median ±1 quartile, with whiskers extending to the most extreme data point within 1.5 interquartile ranges from the box boundaries. (f) Survival analysis of ROR1 and YAP1 high and low expressing breast cancer patients based on expression intensity (Log-rank test; p < .0001, scale bar: 50 μM). (g) Bar graph showing the ROR1 and YAP1 expression intensity (%) in treatment naïve, sensitive, partially sensitive and resistant patient tumors (**p < .0, two-tailed Student's t -test).

Article Snippet: Human ROR1 (AF2000, R&D System, USA), HER2 (MAB1129, R & D System, USA) and YAP1(MAB8094, R & D System, USA) antibodies were purchased from R & D System, USA.

Techniques: Over Expression, Mutagenesis, Expressing, Immunohistochemical staining, Two Tailed Test

Journal: EBioMedicine

Article Title: Antibody-drug conjugate T-DM1 treatment for HER2+ breast cancer induces ROR1 and confers resistance through activation of Hippo transcriptional coactivator YAP1

doi: 10.1016/j.ebiom.2019.04.061

Figure Lengend Snippet: Treatment resistant BC cells overexpress ROR1 have a higher sphere forming efficiency: (a) Administration of therapy leads to the increase of ROR1 expression intensity in HER2 + treatment naïve and treatment exposed BC patient ( p = 3.021e-02. unpaired two-sided t -test). (b) Tumor cells were dissociated from treatment naïve, sensitive and resistant HER2 + BC patients and equal numbers of cells were cultured in the supplemented CSC medium and stained for ROR1 expression in the spheres. Matched HER2 + BC patient's tissues were stained for ROR1 expression by immunofluorescence (Scale bar: 50 μM). (c) FACS analysis of ROR1 + and ROR1 − population in treatment naïve, sensitive and resistant cells from primary HER2 + BC patient tumors. (d) Equal number of sorted ROR1 + and ROR1 − cells from primary BC patients ( n = 2) were cultured in the supplemented CSC medium and analyzed for sphere forming efficiency ( p < .0002, paired two-sided t -test). (e) Comparison of epithelial-to-mesenchymal transition related genes showing mRNA expression from treatment naïve, sensitive and resistant ROR1 + and ROR1 − primary HER2 + BC patients cell fractions (results are mean fold change +/− SE, n = 3). (f) Comparison of ROR1 mRNA expression in primary HER2 + treatment naïve, sensitive and resistant BC patient tumors (results are mean fold change +/− SE, n = 3). (g) In vitro treatment of HER2 + primary tumors cells treated with T-DM1 (5 nM) and assessed for sphere forming efficiency (black arrowhead). Below is shown immunoblotting of ROR1 from each group of patients' protein samples (results are mean +/− SE, n = 3 run in triplicate; Scale bar: 100 μM).

Article Snippet: Human ROR1 (AF2000, R&D System, USA), HER2 (MAB1129, R & D System, USA) and YAP1(MAB8094, R & D System, USA) antibodies were purchased from R & D System, USA.

Techniques: Expressing, Cell Culture, Staining, Immunofluorescence, Comparison, In Vitro, Western Blot

Journal: EBioMedicine

Article Title: Antibody-drug conjugate T-DM1 treatment for HER2+ breast cancer induces ROR1 and confers resistance through activation of Hippo transcriptional coactivator YAP1

doi: 10.1016/j.ebiom.2019.04.061

Figure Lengend Snippet: T-DM1 treatment alters ROR1 expression without significant changes in HER2 expression: (a) Immunostaining of HER2 expression from treatment naïve ( n = 5), sensitive (n = 5) and resistant (n = 5) HER2 + BC patient's tumor samples (Scale bar: 50 μM). (b) HCC1954 and MCF-7-HER2+ cells were cultured on a glass cover slip in 24-well plates and treated either with vehicle (DMSO) or 5 nM of T-DM1 for 5 days. Cells were fixed and immunofluorescence stained images captured for HER2 (red) and nucleus DAPI (blue) (Scale bar: 50 μM). (c) HCC1954 and MCF-7-HER2+ cells were cultured, treated and fixed as (b), and immunofluorescently stained for ROR1 (red) and nuclear DAPI (blue) expression (Scale bar: 50 μM). (d) Treatment naïve BC patient tumor cells, (e) HCC1954(HER2+), and (f) MCF-7-HER2+ cells were treated with T-DM1 (5 nM) for 5 days, trypsinized and cultured in supplemented CSC medium in sphere culture for 10 days (10 days is denoted as first generation). After 10 days, spheres were dissociated and re-cultured in CSC medium for an additional 10 days (20 days denoted as second generation) followed by dissociation of spheres and then re-cultured again in CSC medium (30 days denoted as third generation). In every 10-day culture cycle, sphere forming efficiency was examined in ROR1 + and ROR1 − populations by counting the number and size of spheres. (Bar graph represents the mean +/− SE, n = 3; run in triplicate; p = ns [not significant]; p = .05; p = .01, p = .001; one-way ANOVA). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Human ROR1 (AF2000, R&D System, USA), HER2 (MAB1129, R & D System, USA) and YAP1(MAB8094, R & D System, USA) antibodies were purchased from R & D System, USA.

Techniques: Expressing, Immunostaining, Cell Culture, Immunofluorescence, Staining

Journal: EBioMedicine

Article Title: Antibody-drug conjugate T-DM1 treatment for HER2+ breast cancer induces ROR1 and confers resistance through activation of Hippo transcriptional coactivator YAP1

doi: 10.1016/j.ebiom.2019.04.061

Figure Lengend Snippet: T-DM1 treatment-induced ROR1 + cells enriched with CSC features show higher self-renewal efficiency and increased resistance with enriched CSC features. (a) Treatment naïve HER2 + BC patients tumor cells were enzymatically dissociated and cultured in supplemented CSC medium, treated with either vehicle (DMSO) and 5 nM T-DM1 and incubated for 10 days (follow the schematic diagram). Spheres were dissociated for single cells and subjected to FACS sorting for ROR1. Extreme limiting dilution assay (ELDA) was performed by plating sorted cells with increasing cell numbers and analyzed for stemness frequencies by ‘R' statistical package “statmod” ( p < .0427; one-way ANOVA). (right) showed the confidence interval for 1/stemness frequency. (b) Bulk cells were isolated and treated as described in (a) and analyzed for sphere forming efficiency (Bar graph represents the mean +/− SE, n = 3, run in triplicate). (c) HER2 + patient tumor cells treated with either vehicle or T-DM1 for 5 days, harvested and FACS sorted for ROR1 + and ROR1 − populations, and then sorted cells were cultured in supplemented CSC medium [follow the schematic diagram] for 10 days and analyzed for the sphere forming efficiency from each cell population (black arrowhead; p = .001 for T-DM1 treatment; p = .05 for vehicle, unpaired two-sided t -test, Scale bar: 100 μM). (d) HCC1954 cells were treated and analyzed as described in (c) [black arrowhead; p = .01 for T-DM1 treatment; p = ns (not significant) for vehicle, unpaired two-sided t -test; Scale bar: 100 μM]. (e) HCC1954 cells were either treated with vehicle or T-DM1 and sorted as described in (c) and quantitatively analyzed for Bmi1, Nanog, Oct3/4. Sox2 mRNA in ROR1 + and ROR1 − populations. (Bar graph represents the mean +/− SE, n = 3; run in triplicate; p = .05; p = .01; p = .001; one-way ANOVA). (f) ROR1 + cells were more significantly resistant than ROR1 − cells at individual T-DM1 treatment and showed higher EC50 in response to T-DM1 as compared to ROR1 − cells. (Bar graph represents the mean +/− SE, n = 3; run in triplicate; p = .05, p = .01).

Article Snippet: Human ROR1 (AF2000, R&D System, USA), HER2 (MAB1129, R & D System, USA) and YAP1(MAB8094, R & D System, USA) antibodies were purchased from R & D System, USA.

Techniques: Cell Culture, Incubation, Limiting Dilution Assay, Isolation

Journal: EBioMedicine

Article Title: Antibody-drug conjugate T-DM1 treatment for HER2+ breast cancer induces ROR1 and confers resistance through activation of Hippo transcriptional coactivator YAP1

doi: 10.1016/j.ebiom.2019.04.061

Figure Lengend Snippet: T-DM1 treatment induced ROR1 + cells are enriched for CD44, ALDH and YAP1 target genes: (a-b) Freshly dissociated treatment naïve and treatment resistant HER2 + BC patients tumor cells were FACS sorted and analyzed for ROR1 subpopulation stained with ALDH and CD44 antibodies (Results are median +/− interquartile range (IQR) n = 5; unpaired two-sided t -test). (c-d. Patients tumor cells were either treated with vehicle (DMSO) and/or T-DM1 for 5 days, and sorted as (a) Analysis of ALDH and CD44 expression by FACS and immunofluorescence staining in ROR1 + and ROR1 − subpopulation (Scale bar: 50 μM). (e) HCC1954 cells treated, maintained, sorted and analyzed as (d). (g) Cells were treated and sorted as (d), extracted total RNA followed by qRT-PCR for YAP1, TEAD1, CTGF and CCND1 in ROR1 + and ROR1 − populations (Bar graph represents the mean +/− SE, n = 3; run in triplicate; p = .001, 0.01, 0.05, 0.001 and 0.001). (h) Detection of CTGF from treatment naïve and treatment exposed patients' blood serum by ELISA assay (Bar graph represents the mean +/− SE, n = 3; run in triplicate; ** p < .01; Students t -test).

Article Snippet: Human ROR1 (AF2000, R&D System, USA), HER2 (MAB1129, R & D System, USA) and YAP1(MAB8094, R & D System, USA) antibodies were purchased from R & D System, USA.

Techniques: Staining, Expressing, Immunofluorescence, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: EBioMedicine

Article Title: Antibody-drug conjugate T-DM1 treatment for HER2+ breast cancer induces ROR1 and confers resistance through activation of Hippo transcriptional coactivator YAP1

doi: 10.1016/j.ebiom.2019.04.061

Figure Lengend Snippet: Silencing ROR1 sensitizes T-DM1 treatment-induced ROR1 + spheroids forming efficiency and tumor initiation: (a) Patients tumor cells, (b) HCC1954, and (c) MCF-7 HER2+ cells. T-DM1 and ROR1-shRNA co-treatment resulted in eradication of sphere forming efficiency. (d-f) Loss of ROR1 expression in the spheres was analyzed by immunofluorescence staining (Scale bar: 50 μM) (red-ROR1, blue-DAPI) in HER2 + BC patients tumor cells, HCC1954 and MCF-7-HER2+ cells (Bar graph represents the mean +/− SE, n = 3; run in triplicate; p = .05; p = .01; one-way ANOVA). (g) Average number of spheres assessed for HCC1954 and MCF-7-HER2+ cells after transfecting with control shRNA and ROR1-shRNA. (h) Representation of tumors grown in mice after transfecting HCC1954 cells with either control-shRNA or ROR-shRNA. Table below indicates the number of tumors initiated in each group with percentage. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Human ROR1 (AF2000, R&D System, USA), HER2 (MAB1129, R & D System, USA) and YAP1(MAB8094, R & D System, USA) antibodies were purchased from R & D System, USA.

Techniques: shRNA, Expressing, Immunofluorescence, Staining, Control

Journal: EBioMedicine

Article Title: Antibody-drug conjugate T-DM1 treatment for HER2+ breast cancer induces ROR1 and confers resistance through activation of Hippo transcriptional coactivator YAP1

doi: 10.1016/j.ebiom.2019.04.061

Figure Lengend Snippet: T-DM1 treatment- induced ROR1 + cells have high tumor forming capacity in vivo : (a) Schematic representation of cell sorting and tumor initiation assay. (b) Fresh HER2 + breast tumor cells (n = 5 patient) were dissociated and FACS sorted ROR1 + and ROR1 − cells as described previously. Equal number of sorted and unsorted bulk cells from each patient tumor were injected subcutaneously in Nu/J mice (n = 5) and tumor growth and incidence were monitored for 8 weeks. ROR1 + cells were capable of generating larger tumors (5 out of 5 tumors), ROR1 − cells (2 out of 5 tumors) and unsorted bulk cells (3 out of 5 tumors) generated tumors but smaller in size (Scale bar 5 mm). (c) The weight distributions of tumors generated from ROR + , ROR1 − and unsorted bulk tumor cells are shown in all patient's tumors. Results are median +/− IQR (Inter quartile range), p = .01 and 0.05; repeated measure ANOVA. (d) HCC1954 cells were either treated with vehicle (DMSO) and/or T-DM1 for 5 days and sorted based on T-DM1 induced cell surface ROR1 overexpression and injected to mice. Tumor growth and incidence were monitored for 8 weeks. On the basis of tumor incidence in the indicated time, a Kaplan-Meier survival curve was generated using Log-rank test (Log-rank P = .03). “R” statistical software “survival” and “survminer” packages were used to analyze the tumor-free survival curve. (e) In vivo limiting dilution assay was performed using HCC1954 cells and treatment and sorting performed as described in (a). Sorted 100, 1000, 10,000 and 100,000 cells were mixed with Matrigel and injected subcutaneously in Nu/J mice (n = 5, Scale bar 5 mm). (f) Tumor initiating capacity was monitored over 8 weeks and CSC frequency was calculated using “R” statistical software “statmod” package. The frequency of tumor-initiating cells for 3 mice is graphed. Results are median +/− IQR (repeated measure ANOVA).

Article Snippet: Human ROR1 (AF2000, R&D System, USA), HER2 (MAB1129, R & D System, USA) and YAP1(MAB8094, R & D System, USA) antibodies were purchased from R & D System, USA.

Techniques: In Vivo, FACS, Injection, Generated, Over Expression, Software, Limiting Dilution Assay

Journal: EBioMedicine

Article Title: Antibody-drug conjugate T-DM1 treatment for HER2+ breast cancer induces ROR1 and confers resistance through activation of Hippo transcriptional coactivator YAP1

doi: 10.1016/j.ebiom.2019.04.061

Figure Lengend Snippet: YAP1 regulates T-DM1 treatment-induced ROR1 overexpression and CSC enrichment. (a) HCC1954 cells were transduced with full length YAP1 cDNA (PIN20YYAP1). Doxycycline (1 μg/ml) was added in the cell culture medium to induce the YAP1 expression. Western blot was performed using antibodies to YAP1, ROR1 and HER2. (b) Immunofluorescence staining for YAP1 (green; white arrow head) and ROR1 (red; white arrowhead) and DAPI (blue) confirming the transduction of YAP1 in the presence and absence of doxycycline (Scale bar: 50 μM). (c) YAP1 was knockdown by two independent YAP1 siRNA and Western Blot was performed to confirm the expression of YAP1 and ROR1 (red label box siRNA was used in our experiments). (d) HCC1954 cells were transfected with non-targeting and YAP1 specific siRNA for 48 h. Cells were then treated with T-DM1 for 48 h, followed by treatment with ROR1-shRNA for 48 h. Treated cells were trypsinized, cultured in the supplemented CSC medium, and assessed for sphere forming efficiency (black arrowhead) Bar graph represents the mean +/− SE, n = 3; run in triplicate; p = .05; p = .01; one-way ANOVA. [Scale bar: 100 μM]. (e) Immunoblots showing the expression of YAP1, ROR1 and HER2 after treatment of HCC1954 cells with 2 μg/mL verteporfin. (f) Quantification of sphere forming efficiency (black arrowhead) after verteporfin treatment at 2 μg/ml and vehicle (DMSO) in HCC1954 cells. Bar graph represents the mean +/− SE, n = 3; run in triplicate; p = .05; p = .01; Student's t -test; Scale bar: 100 μM). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Human ROR1 (AF2000, R&D System, USA), HER2 (MAB1129, R & D System, USA) and YAP1(MAB8094, R & D System, USA) antibodies were purchased from R & D System, USA.

Techniques: Over Expression, Transduction, Cell Culture, Expressing, Western Blot, Immunofluorescence, Staining, Knockdown, Transfection, shRNA

Journal: International Journal of Molecular Sciences

Article Title: Effect of the Protein Corona Formation on Antibody Functionalized Liquid Lipid Nanocarriers

doi: 10.3390/ijms242316759

Figure Lengend Snippet: Schematic representation of the procedure steps for the preparation of LLCNs and subsequent functionalization to obtain immune-nanocapsules (LLNCs-αHER2).

Article Snippet: The monoclonal ErbB-2 antibody anti-human Vio ® Bright FITC (αHER2) (clone 24D2) from humans was obtained by Miltenyi Biotech (Madrid, Spain).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Effect of the Protein Corona Formation on Antibody Functionalized Liquid Lipid Nanocarriers

doi: 10.3390/ijms242316759

Figure Lengend Snippet: Mean diameter, standard deviation (SD), and mode of the LLNCs, LLNCs-αHER2, and LLNCs-αHER2-PC measured at 25 °C with NTA and DLS techniques in pH 7.4 buffer.

Article Snippet: The monoclonal ErbB-2 antibody anti-human Vio ® Bright FITC (αHER2) (clone 24D2) from humans was obtained by Miltenyi Biotech (Madrid, Spain).

Techniques: Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Effect of the Protein Corona Formation on Antibody Functionalized Liquid Lipid Nanocarriers

doi: 10.3390/ijms242316759

Figure Lengend Snippet: Hydrodynamic size distribution of the LLNCs, LLNCs-αHER2, and LLNCs-αHER2-PC measured at 25 °C with NTA technique in pH 7 buffer.

Article Snippet: The monoclonal ErbB-2 antibody anti-human Vio ® Bright FITC (αHER2) (clone 24D2) from humans was obtained by Miltenyi Biotech (Madrid, Spain).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Effect of the Protein Corona Formation on Antibody Functionalized Liquid Lipid Nanocarriers

doi: 10.3390/ijms242316759

Figure Lengend Snippet: Physico-chemical characterization: ( a ) Zeta potential of the LLNCs (▪), LLNCs-αHER2 ( ● ), LLNCs-αHER2-FBS ( ✱ ), and LLNCs-αHER2-PC ( ▷ ) measured at 25 °C as a function of the medium pH and low ionic strength. ( b ) SDS-PAGE analysis under reducing conditions of different LLNCs. (C) Molecular weight marker: (1) αHER2; (2) FBS; (3) Fibrinogen; (4) LLNCs-αHER2; (5) elution volume after cleaning LLNCs-αHER2; (6) LLNCs-αHER2-PC; (7) elution volume after cleaning LLNCs-αHER2-PC.

Article Snippet: The monoclonal ErbB-2 antibody anti-human Vio ® Bright FITC (αHER2) (clone 24D2) from humans was obtained by Miltenyi Biotech (Madrid, Spain).

Techniques: Zeta Potential Analyzer, SDS Page, Molecular Weight, Marker

Journal: International Journal of Molecular Sciences

Article Title: Effect of the Protein Corona Formation on Antibody Functionalized Liquid Lipid Nanocarriers

doi: 10.3390/ijms242316759

Figure Lengend Snippet: LLNCs-αHER2-PC after incubation of immune-nanocapsules in a simulated physiological medium with FBS supplemented with FB.

Article Snippet: The monoclonal ErbB-2 antibody anti-human Vio ® Bright FITC (αHER2) (clone 24D2) from humans was obtained by Miltenyi Biotech (Madrid, Spain).

Techniques: Incubation

Journal: International Journal of Molecular Sciences

Article Title: Effect of the Protein Corona Formation on Antibody Functionalized Liquid Lipid Nanocarriers

doi: 10.3390/ijms242316759

Figure Lengend Snippet: Representative confocal microscopy of ( A ) SKBR3 and ( B ) HDFa (scale bar = 20 μm) incubated for 60 min with NR-LLNCs, NR-LLNCs-HER2, and NR-LLNCs-HER2-PC. Red filter and green filter correspond to LLNCs labeled with Nile Red and LLNCs labeled with HER2-FITC, respectively.

Article Snippet: The monoclonal ErbB-2 antibody anti-human Vio ® Bright FITC (αHER2) (clone 24D2) from humans was obtained by Miltenyi Biotech (Madrid, Spain).

Techniques: Confocal Microscopy, Incubation, Labeling

Journal: International Journal of Molecular Sciences

Article Title: Effect of the Protein Corona Formation on Antibody Functionalized Liquid Lipid Nanocarriers

doi: 10.3390/ijms242316759

Figure Lengend Snippet: In vitro uptake of NR-LLNCs, NR-LLNCs-HER2, and NR-LLNCs-HER2-PC in both SKBR3 and HDFa analyzed by flow cytometry. Values represent the fluorescence intensity of the merge channels for green and red as NR-LLNCs-HER2 are both red and green fluorescent. Data are mean values ± SD. * p < 0.001 shows the significant values calculated using t -test.

Article Snippet: The monoclonal ErbB-2 antibody anti-human Vio ® Bright FITC (αHER2) (clone 24D2) from humans was obtained by Miltenyi Biotech (Madrid, Spain).

Techniques: In Vitro, Flow Cytometry, Fluorescence

Journal: Scientific Reports

Article Title: ELISA assay employing epitope-specific monoclonal antibodies to quantify circulating HER2 with potential application in monitoring cancer patients undergoing therapy with trastuzumab

doi: 10.1038/s41598-020-59630-y

Figure Lengend Snippet: Competitive binding test for mAbs MGR2, MGR3, and trastuzumab toward immobilized HER2 ECD, determined by Biacore X100. ( a ) Trastuzumab (1st injection) vs MGR3 (2nd injection); ( b ) MGR3 (1st injection) vs trastuzumab (2nd injection); ( c ) trastuzumab (1st injection) vs MGR2 (2nd injection); ( d ) MGR2 (1st injection) vs trastuzumab (2nd injection); ( e ) MGR3 (1st injection) vs MGR2 (2nd injection) ( f ) MGR2 (1st injection) vs MGR3 (2nd injection).

Article Snippet: Goat anti human HER2 biotinylated polyclonal antibody (IgG pAb-biotin, code BAF1129) and Streptavidin-HRP conjugate (code DY998 Part #890803 5 vials) were from R&D Systems and stored at −20 °C and at 4 °C respectively.

Techniques: Binding Assay, Injection

Journal: Scientific Reports

Article Title: ELISA assay employing epitope-specific monoclonal antibodies to quantify circulating HER2 with potential application in monitoring cancer patients undergoing therapy with trastuzumab

doi: 10.1038/s41598-020-59630-y

Figure Lengend Snippet: SPR sensograms showing the kinetic analysis performed on HER2 ECD antigen immobilized on CM5 chip. mAbs were tested at the following concentrations: ( a ) 0.25–30 nM for trastuzumab; ( b ) 1–75 nM for MGR2; ( c ) 2.5–200 nM for MGR3. Association time 500 s, dissociation time 400 s.

Article Snippet: Goat anti human HER2 biotinylated polyclonal antibody (IgG pAb-biotin, code BAF1129) and Streptavidin-HRP conjugate (code DY998 Part #890803 5 vials) were from R&D Systems and stored at −20 °C and at 4 °C respectively.

Techniques:

Journal: Scientific Reports

Article Title: ELISA assay employing epitope-specific monoclonal antibodies to quantify circulating HER2 with potential application in monitoring cancer patients undergoing therapy with trastuzumab

doi: 10.1038/s41598-020-59630-y

Figure Lengend Snippet: ELISA standard curve for HER2 ECD quantification in the range 0–12.5 ng mL −1 , employing ( a ) 2 µg mL −1 MGR2 or ( b ) 5 µg mL −1 MGR3 mAbs as the capture antibodies and anti-HER2 ECD pAb as detection antibody. Error bars indicate standard deviation (SD).

Article Snippet: Goat anti human HER2 biotinylated polyclonal antibody (IgG pAb-biotin, code BAF1129) and Streptavidin-HRP conjugate (code DY998 Part #890803 5 vials) were from R&D Systems and stored at −20 °C and at 4 °C respectively.

Techniques: Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Scientific Reports

Article Title: ELISA assay employing epitope-specific monoclonal antibodies to quantify circulating HER2 with potential application in monitoring cancer patients undergoing therapy with trastuzumab

doi: 10.1038/s41598-020-59630-y

Figure Lengend Snippet: ELISA standard curve for HER2 ECD quantification in the range 0.19–12.5 ng mL −1 , employing 5 µg mL −1 MGR3 as the capture antibody and 15 µg mL −1 biotinylated MGR2 as detector mAb. Error bars indicate SD.

Article Snippet: Goat anti human HER2 biotinylated polyclonal antibody (IgG pAb-biotin, code BAF1129) and Streptavidin-HRP conjugate (code DY998 Part #890803 5 vials) were from R&D Systems and stored at −20 °C and at 4 °C respectively.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: ELISA assay employing epitope-specific monoclonal antibodies to quantify circulating HER2 with potential application in monitoring cancer patients undergoing therapy with trastuzumab

doi: 10.1038/s41598-020-59630-y

Figure Lengend Snippet: Linearity of quantitative measurement procedure represented as percentage recovery (Expected/Observed HER2 ECD level) for different proportions SL (Serum Low) and SH (Serum High).

Article Snippet: Goat anti human HER2 biotinylated polyclonal antibody (IgG pAb-biotin, code BAF1129) and Streptavidin-HRP conjugate (code DY998 Part #890803 5 vials) were from R&D Systems and stored at −20 °C and at 4 °C respectively.

Techniques:

Journal: Scientific Reports

Article Title: ELISA assay employing epitope-specific monoclonal antibodies to quantify circulating HER2 with potential application in monitoring cancer patients undergoing therapy with trastuzumab

doi: 10.1038/s41598-020-59630-y

Figure Lengend Snippet: Bland-Altman plot of differences between ( a ) ADVIA Centaur or ( b ) Quantikine HER2 ELISA kit and in house developed ELISA. Passing-Bablok regression analysis (n = 11 serum samples) comparing ( c ) ADVIA Centaur or ( d ) Quantikine HER2 ELISA kit with the in-house ELISA. In Bland-Altman analysis (panels a and b), the difference between the tested methods is plotted against the mean for HER2 ECD level. Black dotted lines show limits of agreement (95% CI) and the blue line shows the mean value of the differences between the two methods under comparison (the bias). The red dotted line is the zero line for assessing the discrepancy of the observed mean difference from zero. In Passing–Bablok agreement analyses (panels c and d), the data obtained with the in-house ELISA (y axis) is plotted against those of the commercial reference methods (x axis) together with the identity line (y = x) and the regression line. The regression line is continuous and blue, the red dotted line is the identity line (y = x) and the black dashed lines are the CI. Results are summarised as it follows. ( c ) regression equation, y = 1.41 + 0.87x; intercept, 1.41 (95% CI −5.85 to 7.39); slope, 0.87 (95% CI 0.33 to 1.56). ( d ) regression equation, y = −0.37 + 1.08x; intercept, −0.37 (95% CI −3.17 to 2.86); slope, 1.08 (95% CI 0.72 to 1.40).

Article Snippet: Goat anti human HER2 biotinylated polyclonal antibody (IgG pAb-biotin, code BAF1129) and Streptavidin-HRP conjugate (code DY998 Part #890803 5 vials) were from R&D Systems and stored at −20 °C and at 4 °C respectively.

Techniques: Enzyme-linked Immunosorbent Assay, Comparison

Journal: Scientific Reports

Article Title: ELISA assay employing epitope-specific monoclonal antibodies to quantify circulating HER2 with potential application in monitoring cancer patients undergoing therapy with trastuzumab

doi: 10.1038/s41598-020-59630-y

Figure Lengend Snippet: Determination of HER2 ECD only (black dots) or pre-incubated with trastuzumab at 50 (red triangles) and 200 (blue squares) µg mL −1 employing ( a ) MGR2- or ( b ) MGR3-based assay and ( c ) the dual mAbs sandwich ELISA with MGR3 in coating and biotinylated MGR2 as the detection antibody. Error bars indicate SD but are too small to be visible.

Article Snippet: Goat anti human HER2 biotinylated polyclonal antibody (IgG pAb-biotin, code BAF1129) and Streptavidin-HRP conjugate (code DY998 Part #890803 5 vials) were from R&D Systems and stored at −20 °C and at 4 °C respectively.

Techniques: Incubation, Sandwich ELISA

Journal: Scientific Reports

Article Title: ELISA assay employing epitope-specific monoclonal antibodies to quantify circulating HER2 with potential application in monitoring cancer patients undergoing therapy with trastuzumab

doi: 10.1038/s41598-020-59630-y

Figure Lengend Snippet: Anti HER2 ECD mAbs epitope binding pattern evaluation.

Article Snippet: Goat anti human HER2 biotinylated polyclonal antibody (IgG pAb-biotin, code BAF1129) and Streptavidin-HRP conjugate (code DY998 Part #890803 5 vials) were from R&D Systems and stored at −20 °C and at 4 °C respectively.

Techniques: Binding Assay, Injection