Journal: PLoS ONE

Article Title: Evaluation of a New Recombinant Oncolytic Vaccinia Virus Strain GLV-5b451 for Feline Mammary Carcinoma Therapy

doi: 10.1371/journal.pone.0104337

Figure Lengend Snippet: Western blot analysis of GLV-5b451-, LIVP 6.1.1-infected (MOI of 1.0) or uninfected DT09/06 cells. Protein fractions from cell lysates were isolated at different time points and separated by SDS/PAGE. Western blot analysis was performed as described in material and methods. GLAF-2: anti-VEGF scAb; VVA: Vaccinia virus specific proteins; M: PageRuler Prestained Protein Ladder # 26616 (Thermo Scientific, Bonn, Germany), a mixture of ten blue-, orange-, and green-stained recombinant proteins (10 to 170 kDa), was used as size standards in Western blotting.

Article Snippet: Concentrations of VEGF were determined by VEGF ELISA kit (Thermo Scientific, Rockford, USA) developed for detection of human VEGF (cross-reacts approximately 82% to recombinant feline VEGF; R&D Systems, Inc., catalog number DVE00, page 11, www.RnDSystem.com ), in accordance with the manufacturer's directions.

Techniques: Western Blot, Infection, Isolation, SDS Page, Virus, Staining, Recombinant

Journal: PLoS ONE

Article Title: Evaluation of a New Recombinant Oncolytic Vaccinia Virus Strain GLV-5b451 for Feline Mammary Carcinoma Therapy

doi: 10.1371/journal.pone.0104337

Figure Lengend Snippet: Affinity and cross reactivity of GLAF-2 was demonstrated by ELISA. Equal concentrations of feline, murine, or human VEGF (100 ng/well) were coated on ELISA plates. Seven two-fold dilutions of purified GLAF-2 protein ranging from 2000 ng/ml to 31.3 ng/ml were incubated with feline, murine and human VEGFs. PBS was used as negative control. For further ELISA experimental conditions see material and methods. ODs obtained for various conc. of GLAF-2 against feline, murine and human VEGF were plotted. ELISA was repeated in three independent experiments. Each value represents the mean (n = 3) +/− standard deviations (SD).

Article Snippet: Concentrations of VEGF were determined by VEGF ELISA kit (Thermo Scientific, Rockford, USA) developed for detection of human VEGF (cross-reacts approximately 82% to recombinant feline VEGF; R&D Systems, Inc., catalog number DVE00, page 11, www.RnDSystem.com ), in accordance with the manufacturer's directions.

Techniques: Enzyme-linked Immunosorbent Assay, Purification, Incubation, Negative Control

Journal: PLoS ONE

Article Title: Evaluation of a New Recombinant Oncolytic Vaccinia Virus Strain GLV-5b451 for Feline Mammary Carcinoma Therapy

doi: 10.1371/journal.pone.0104337

Figure Lengend Snippet: ( A ) Western blot analysis of DT09/06 tumor xenografts injected with LIVP 6.1.1 or GLV-5b451 virus (n = 3). The presence of GLAF-2 proteins was performed as described before. Each sample represents an equivalent of 2 mg tumor mass. ( B ) Levels of functional VEGF in tumor lysates determined by ELISA. The graph was plotted using the mean values of each group of three independent measurements. The data are presented as mean values +/− SD. An unpaired t-test was performed revealing significant differences (****P<0.0001, **P<0.01).

Article Snippet: Concentrations of VEGF were determined by VEGF ELISA kit (Thermo Scientific, Rockford, USA) developed for detection of human VEGF (cross-reacts approximately 82% to recombinant feline VEGF; R&D Systems, Inc., catalog number DVE00, page 11, www.RnDSystem.com ), in accordance with the manufacturer's directions.

Techniques: Western Blot, Injection, Virus, Functional Assay, Enzyme-linked Immunosorbent Assay

Journal: Pharmaceutics

Article Title: Elovanoids Counteract Inflammatory Signaling, Autophagy, Endoplasmic Reticulum Stress, and Senescence Gene Programming in Human Nasal Epithelial Cells Exposed to Allergens

doi: 10.3390/pharmaceutics14010113

Figure Lengend Snippet: ELVN-34 reduce the expression of pro-inflammatory cytokines, chemokines and cell adhesion molecule in HNEpC challenged with LPS or poly(I:C). HNEpC challenged with LPS or poly(I:C) (30 μg/mL) display enhanced production of pro-inflammatory cytokines and chemokines IL-6, IL-1β, IL-8/CXCL8, CCL2/MCP-1, CXCL1/KC/GRO, VEGF, and cell adhesion molecule ICAM1(CD54) compared to non-treated cells. This increase in the production of pro-inflammatory molecules is abrogated by the addition of ELVN-34 (500 nM) 30 min post-challenge. LPS and poly(I:C) induce a reduction of anti-inflammatory IL-10 expression in HNEpC that is restored to normal levels after treatment with ELVN-34 (500 nM). The results showed the averages of three independent experiments. (**** p < 0.0001, NS—not significant).

Article Snippet: In addition, the expression of the following cytokines was analyzed in the supernatant of HNEpC exposed to different allergic inductors: IL-6 (Catalog# 6802, Chondrex) range of detection: (9–600 pg/mL), IL-1β (Catalog# 6805, Chondrex) range of detection: (4–250 pg/mL) IL-8/CXCL8 (Quantikine ELISA Kit, Catalog# D8000C, R&D Systems) range of detection: (31–2000 pg/mL), vascular endothelial growth factor (VEGF) (Catalog# 6810, Chondrex) range of detection: (31–2000 pg/mL), ICAM1(CD54) (ELISA Kit, Catalog# ab100640, Abcam) range of detection: (16–1000 pg/mL), CXCL1/KC/GRO (Catalog# 6825, Chondrex) range of detection: CCL2/MCP-1 (Catalog# 6821, Chondrex) range of detection: (16–1000 pg/mL), and IL-10 (Catalog# 6806, Chondrex) range of detection: (8–500 pg/mL).

Techniques: Expressing

Journal: BMC Complementary and Alternative Medicine

Article Title: Astragalus saponins downregulate vascular endothelial growth factor under cobalt chloride-stimulated hypoxia in colon cancer cells

doi: 10.1186/1472-6882-12-160

Figure Lengend Snippet: AST downregulates pro-angiogenic factors in HCT 116 colon cancer cells. ( A ) HCT 116 cells were treated with AST (80 μg/ml) and Western blotting was performed to analyze the level of bFGF and VEGF in a 72-hour time course study. ( B ) Various concentrations of AST (0, 40, 60 or 80 μg/ml) were administered to HCT 116 cells for 24, 48 or 72 h to determine its concentration-dependent effect on bFGF and VEGF level. Representative immunoblots from at least three independent experiments are shown. Relative band densities were summerized in histograms after normalization by β-actin. Arbitrary data are expressed as mean ± S.E.M., with statistical significance * p < 0.05, ** p < 0.01 when compared with the untreated group at 0 h ( A ) or the respective control group ( B ).

Article Snippet: The serum was assayed by using a mouse ELISA kit for detecting VEGF (Calbiochem) according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA).

Techniques: Western Blot, Concentration Assay, Control

Journal: BMC Complementary and Alternative Medicine

Article Title: Astragalus saponins downregulate vascular endothelial growth factor under cobalt chloride-stimulated hypoxia in colon cancer cells

doi: 10.1186/1472-6882-12-160

Figure Lengend Snippet: AST acts by modulation of PI3K/Akt/mTOR signaling but does not involve the MAPK pathway. HCT 116 cells were treated with AST (80 μg/ml) and Western blotting was performed to analyze the level of ( A ) PTEN, p-Akt, Akt, p-mTOR, mTOR and ( B ) p-JNK, JNK, p-p38 and p38 in a 72-hour time course study. ( C ) Combined effects of AST and rapamycin on the pro-angiogenic factors bFGF and VEGF. HCT 116 cells were incubated with AST (80 μg/ml) either in the absence or presence of the mTOR inhibitor rapamycin for 72 h. Rapamycin (50 nM) was added 30 min prior to the AST treatment. Representative immunoblots from at least three independent experiments are shown. β-actin was used as internal control in immunoblot assay. Arbitrary data are expressed as mean ± S.E.M., with statistical significance * p < 0.05, ** p < 0.01 when compared with the untreated group at 0 h ( A ) or control group ( B ); + p < 0.05 when compared with rapamycin group.

Article Snippet: The serum was assayed by using a mouse ELISA kit for detecting VEGF (Calbiochem) according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA).

Techniques: Western Blot, Incubation, Control

Journal: BMC Complementary and Alternative Medicine

Article Title: Astragalus saponins downregulate vascular endothelial growth factor under cobalt chloride-stimulated hypoxia in colon cancer cells

doi: 10.1186/1472-6882-12-160

Figure Lengend Snippet: AST suppresses HIF-1α and VEGF induction under hypoxia in colon cancer cells. CoCl 2 (100 μM) was added to the culture medium of HCT 116 cells 30 min prior to various drug treatments to mimic hypoxic condition. ( A ) The induction of HIF-1α and its downstream molecule VEGF by CoCl 2 over a 24-hour course was monitored in HCT 116 cells. The effect of AST (80 μg/ml) on HIF-1α and VEGF protein and mRNA expression was then assessed. ( B ) Combined treatment of AST and rapamycin (50 nM) suppressed HIF-1α and VEGF protein expression under CoCl 2 -mimicked hypoxic condition in HCT 116 cells. Data represents the mean of at least three independent experiments. β-actin and GAPDH were used as internal control in immunoblot assay and RT-PCR, respectively. Arbitrary data are expressed as mean ± S.E.M., with statistical significance * p < 0.05 when compared with control group; # p < 0.05, ## p < 0.01 when compared with CoCl 2 hypoxia group; + p < 0.05 when compared with CoCl 2 + rapamycin group.

Article Snippet: The serum was assayed by using a mouse ELISA kit for detecting VEGF (Calbiochem) according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA).

Techniques: Expressing, Control, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: BMC Complementary and Alternative Medicine

Article Title: Astragalus saponins downregulate vascular endothelial growth factor under cobalt chloride-stimulated hypoxia in colon cancer cells

doi: 10.1186/1472-6882-12-160

Figure Lengend Snippet: AST suppresses COX-2 and VEGF induction under normoxia and hypoxia in colon cancer cells. ( A ) Effects of AST alone { A } (60 μg/ml) or in combined treatment with indomethacin {A + I} on the level of COX-2 in HT-29 cells under normoxia. ( B ) Combined treatment of AST (80 μg/ml) and indomethacin (100 μM) suppressed the induced COX-2 and VEGF level under CoCl 2 -mimicked hypoxic condition in HCT 116 cells. Data represents the mean of at least three independent experiments. β-actin was used as internal control in immunoblot assay. Arbitrary data are expressed as mean ± S.E.M., with statistical significance * p < 0.05, ** p < 0.01 when compared with the untreated group at 0 h or control group; # p < 0.05, ## p < 0.01 when compared with CoCl 2 hypoxia group; + p < 0.05 when compared with indomethacin (A) or CoCl 2 + indomethacin ( B ) group.

Article Snippet: The serum was assayed by using a mouse ELISA kit for detecting VEGF (Calbiochem) according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA).

Techniques: Control, Western Blot

Journal: BMC Complementary and Alternative Medicine

Article Title: Astragalus saponins downregulate vascular endothelial growth factor under cobalt chloride-stimulated hypoxia in colon cancer cells

doi: 10.1186/1472-6882-12-160

Figure Lengend Snippet: AST inhibits tumor growth and suppresses tissue VEGF level by modulating Akt/mTOR signaling and COX-2. HCT 116 colon cancer cells (2 × 10 6 ) were injected subcutaneously to the Balb/c nu/nu mice. AST (100 mg/kg) was administrated orally once daily for 14 days after the tumors became palpable at Day 10. Tumors from the xenografted mice were excised at the day of sacrifice. ( A ) Tumor volume and ( B ) serum VEGF level in the control and AST-treated groups of animals were measured. Data are expressed as mean ± S.E.M. (n = 6), with statistical significance * p < 0.05, ** p < 0.01 when compared with control group. ( C ) Tissue level of p-Akt, Akt, p-mTOR, mTOR, and ( D ) the pro-angiogenic factors VEGF, VEGFR1 and VEGFR2 was examined in untreated control (#1-2) and AST-treated (#3-4) groups of animals. Representative immunoblots from tumor tissues obtained from six animals in each group are shown. β-actin was used as internal control in immunoblot assay. ( E ) Histological examination of COX-2 level in tissue sections of HCT 116 xenografted nude mice with or without AST treatment was performed. Representative microphotographs of control and drug-treated groups are shown (200x magnification).

Article Snippet: The serum was assayed by using a mouse ELISA kit for detecting VEGF (Calbiochem) according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA).

Techniques: Injection, Control, Western Blot

Journal: Circulation

Article Title: Collagen-Targeting Vascular Endothelial Growth Factor Improves Cardiac Performance After Myocardial Infarction

doi: 10.1161/circulationaha.108.800565

Figure Lengend Snippet: Figure 1. Collagen-binding ability of VEGF and CBD-VEGF in vitro. A, The amount of VEGF or CBD-VEGF bound to collagen was mea- sured by ELISA assay (n6 in each group/dose). Data are presented as meanSEM. *P0.05; **P0.01. B, Kd values for binding of VEGF or CBD-VEGF to collagen were calculated by Scatchard analysis. Slope of each line1/Kd.

Article Snippet: The remaining proteins on collagen were detected with an anti-VEGF monoclonal antibody (1:500, Santa Cruz Biotechnology, Santa Cruz, Calif).

Techniques: Binding Assay, In Vitro, Enzyme-linked Immunosorbent Assay

Journal: Circulation

Article Title: Collagen-Targeting Vascular Endothelial Growth Factor Improves Cardiac Performance After Myocardial Infarction

doi: 10.1161/circulationaha.108.800565

Figure Lengend Snippet: Figure 2. Biological activity of growth factors on collagen. A, HUVEC growth–promoting activity was detected by MTT assay, which resulted in dose-response curves (n6 in each group/dose). B, Collagen-coated plates were washed extensively after incubation with VEGF or CBD-VEGF. Next, the mitogenic activities of the proteins that remained on HUVECs were determined by MTT assay (n6 in each group/dose). Data are presented as meanSEM. **P0.01.

Article Snippet: The remaining proteins on collagen were detected with an anti-VEGF monoclonal antibody (1:500, Santa Cruz Biotechnology, Santa Cruz, Calif).

Techniques: Activity Assay, MTT Assay, Incubation

Journal: Circulation

Article Title: Collagen-Targeting Vascular Endothelial Growth Factor Improves Cardiac Performance After Myocardial Infarction

doi: 10.1161/circulationaha.108.800565

Figure Lengend Snippet: Figure 3. VEGF and CBD-VEGF induced blood vessel formation in collagen mem- branes implanted subcutaneously. A, Macroscopic view of the implants retrieved 2 weeks later. Scale bar5 mm. B through D, Immunohistochemical stain- ing of von Willebrand factor on collagen membranes loaded with PBS (B), VEGF (C), or CBD-VEGF (D). Arrows point to typical blood vessels. E, Blood vessel density is expressed as the number of blood vessels per field (n5 in each group). Data are presented as meanSEM *P0.05, **P0.01. Scale bar50 m.

Article Snippet: The remaining proteins on collagen were detected with an anti-VEGF monoclonal antibody (1:500, Santa Cruz Biotechnology, Santa Cruz, Calif).

Techniques: Immunohistochemical staining, Staining

Journal: Circulation

Article Title: Collagen-Targeting Vascular Endothelial Growth Factor Improves Cardiac Performance After Myocardial Infarction

doi: 10.1161/circulationaha.108.800565

Figure Lengend Snippet: Figure 4. Binding ability of VEGF and CBD-VEGF to the infarcted heart. A, After ligation, saline, VEGF, or CBD-VEGF was injected into 5 regions in the border zone surrounding the infarct. Black dots indicate injection sites. B, Three hours after injection, exogenous pro- tein levels were detected by Western blot with anti-polyhistidine antibody. -Actin was used as internal control. C, Quantitation of pro- tein bands (n4 in each group). D, Exogenous protein levels in serum were detected 3 and 6 hours after injection (n6 in each group). Data are presented as meanSEM. *P0.05, **P0.01.

Article Snippet: The remaining proteins on collagen were detected with an anti-VEGF monoclonal antibody (1:500, Santa Cruz Biotechnology, Santa Cruz, Calif).

Techniques: Binding Assay, Ligation, Saline, Injection, Western Blot, Control, Quantitation Assay

Journal: Circulation

Article Title: Collagen-Targeting Vascular Endothelial Growth Factor Improves Cardiac Performance After Myocardial Infarction

doi: 10.1161/circulationaha.108.800565

Figure Lengend Snippet: Figure 5. Histological analysis of hearts 4 weeks after myocardial infarction. Heart sections were stained with Masson trichrome. A, Control group. B, VEGF group. C, CBD-VEGF group. D, Percentage of infarct area in the whole LV area. E, Wall thickness of infarct area. n7 in each group. Data are presented as meanSEM. *P0.05. Scale bar1 mm.

Article Snippet: The remaining proteins on collagen were detected with an anti-VEGF monoclonal antibody (1:500, Santa Cruz Biotechnology, Santa Cruz, Calif).

Techniques: Staining, Control

Journal: Circulation

Article Title: Collagen-Targeting Vascular Endothelial Growth Factor Improves Cardiac Performance After Myocardial Infarction

doi: 10.1161/circulationaha.108.800565

Figure Lengend Snippet: Figure 6. Capillary density in the infarct border zone. Von Wille- brand factor antibody was used in immunohistochemistry of blood vessels. A, control group. B, VEGF group. C, CBD-VEGF group. D, Quantitative analysis of capillary density. Capillary density is expressed as the number of capillary vessels per field. n7 in each group. Data are presented as meanSEM. *P0.05, **P0.01. Scale bar50 m.

Article Snippet: The remaining proteins on collagen were detected with an anti-VEGF monoclonal antibody (1:500, Santa Cruz Biotechnology, Santa Cruz, Calif).

Techniques: Immunohistochemistry, Control

Journal: BioMed Research International

Article Title: Rapid Production of Human VEGF-A following Intradermal Injection of Modified VEGF-A mRNA Demonstrated by Cutaneous Microdialysis in the Rabbit and Pig In Vivo

doi: 10.1155/2019/3915851

Figure Lengend Snippet: Placement of VEGF-A mRNA injections in relation to the microdialysis probe in the rabbit study. For further details, see methods.

Article Snippet: An Alexa-labelled detection antibody against human VEGF-A (R&D systems) was then flowed through the column and the florescence intensity was used for quantification of the ligand.

Techniques:

Journal: BioMed Research International

Article Title: Rapid Production of Human VEGF-A following Intradermal Injection of Modified VEGF-A mRNA Demonstrated by Cutaneous Microdialysis in the Rabbit and Pig In Vivo

doi: 10.1155/2019/3915851

Figure Lengend Snippet: Placement of VEGF-A mRNA injections in relation to the microdialysis probe in the pig study. For further details, see methods.

Article Snippet: An Alexa-labelled detection antibody against human VEGF-A (R&D systems) was then flowed through the column and the florescence intensity was used for quantification of the ligand.

Techniques:

Journal: BioMed Research International

Article Title: Rapid Production of Human VEGF-A following Intradermal Injection of Modified VEGF-A mRNA Demonstrated by Cutaneous Microdialysis in the Rabbit and Pig In Vivo

doi: 10.1155/2019/3915851

Figure Lengend Snippet: Concentrations of human VEGF-A in eluates from 100 kDa microdialysis probes intradermally inserted in the rabbit hind leg. Microdialysis was started at t=0 h and four id injections of VEGF-A mRNA injections (50 μ g each) were given at t=1 h. Dotted line indicates Lower Limit of Quantification (LLOQ, 33.4 pg/mL). Two probes were inserted in each rabbit. Values shown are mean ± SEM (n=4).

Article Snippet: An Alexa-labelled detection antibody against human VEGF-A (R&D systems) was then flowed through the column and the florescence intensity was used for quantification of the ligand.

Techniques:

Journal: BioMed Research International

Article Title: Rapid Production of Human VEGF-A following Intradermal Injection of Modified VEGF-A mRNA Demonstrated by Cutaneous Microdialysis in the Rabbit and Pig In Vivo

doi: 10.1155/2019/3915851

Figure Lengend Snippet: Concentrations of human VEGF-A in microdialysis eluates from 100 kDa microdialysis probes intradermally inserted on the pig (n=3) abdomen. Values presented as individual values in eluate from each probe. Six intradermal VEGF-A mRNA injections (50 μ L each, total 24, 120, or 600 μ g VEGF-A mRNA) were given at t=0 h. At t=1.5 h after the injections, microdialysis was started. Eluates were collected during the time intervals from 1.5 h to 3.5 h, from 3.5 h to 5.5 h, and from 5.5 h to 7.5 h, respectively. Dotted vertical line indicates Lower Limit of Quantification (33.4 pg/mL); eluate samples below LLOQ are depicted as 0.5 ∗ LLOQ=16.7 pg/mL. Six probes were inserted in each pig, two probes per dose except the 120 μ g dose, where only four probes were used to assess human VEGF-A production following injection of 24 or 600 μ g VEGF-A mRNA. The remaining two probes were used to study effects following injection of citrate/saline vehicle.

Article Snippet: An Alexa-labelled detection antibody against human VEGF-A (R&D systems) was then flowed through the column and the florescence intensity was used for quantification of the ligand.

Techniques: Injection, Saline

Journal: BioMed Research International

Article Title: Rapid Production of Human VEGF-A following Intradermal Injection of Modified VEGF-A mRNA Demonstrated by Cutaneous Microdialysis in the Rabbit and Pig In Vivo

doi: 10.1155/2019/3915851

Figure Lengend Snippet: Amounts (pg/mg) of human VEGF-A in skin biopsies excised approximately 8 hours after intradermal injection of VEGF-A mRNA in 3 pigs. Each dose was administered as 6 (24 and 600 μ g dose) or 4 (120 μ g dose) separate injections at two different sites in each pig. Hatched data point in the 24 μ g dose, depicted at 0.1 pg/mg, indicating amount below Lower Limit of Quantification (0.28 pg/mg).

Article Snippet: An Alexa-labelled detection antibody against human VEGF-A (R&D systems) was then flowed through the column and the florescence intensity was used for quantification of the ligand.

Techniques: Injection

Journal: Nature Communications

Article Title: Selective targeting of ligand-dependent and -independent signaling by GPCR conformation-specific anti-US28 intrabodies

doi: 10.1038/s41467-021-24574-y

Figure Lengend Snippet: a Overview of biotinylation of proximal proteins by US28-Bio ID2. b US28-mediated inositol phosphate (IP) accumulation in HEK293T cells expressing HA-US28 wildtype (US28 WT) or HA-US28-Bio ID2(No Nb) alone or co-expressed with non-US28 targeting intrabody-mVenus (Irr Nb mV) or VUN103-mVenus (VUN103 mV). IP levels were normalized to US28 WT No Nbt ( n = 3 independent experiments). c Detection of biotinylated proteins in lysates from HEK293T cells with and without expression of HA-US28-Bio ID2 alone (No Nb) or withIrr Nb mV or VUN103 mV by Western blot using streptavidin-HRP. Representative blot shown from three independent biological replicates. d Detection of pyruvate carboxylase (PC), intrabody-mVenus (mVenus), Gα q and actin in lysates and biotinylated protein pulldown samples of HEK293T cells with or without HA-US28-Bio ID2 alone (No Nb) or withVUN103 mV or Irr Nb mV. Representative blot shown from three independent biological replicates. Samples are derived from the same experiment and blots were processed in parallel. e Quantification of biotinylated Gα q protein levels of biological triplicate samples. Data from three independent experiments using independent biological replicates. f BRET between US28-Nluc and Venus-mini-Gα q upon inducible expression of non-US28 targeting intrabody (Irr Nb-FLAG) VUN103 (VUN103-FLAG)(Intrabody). BRET signals were normalized to that in non-induced HEK293T cells (No intrabody) ( n = 3 independent experiments). g Detection of PC, mVenus, β-arrestin1/2 (β−Arr 1/2), and 14-3-3 in lysates and biotinylated protein pulldown samples of HEK293T cells with or without HA-US28-Bio ID2 alone (No Nb) or with VUN103 mV or Irr Nb mV. Representative blot shown from three independent biological replicates. Samples are derived from the same experiment and blots were processed in parallel. h Quantification of biotinylated βArr-1/2 protein levels in biological triplicate samples. Data from three independent experiments using independent biological replicates. i BRET between US28-Nluc and β-arrestin2-mVenus upon inducible expression of Irr Nb-FLAG or VUN103-FLAG(Intrabody). BRET signals were normalized to that in non-induced HEK293T cells (No intrabody) ( n = 3 independent experiments). All data are plotted as mean ± SD. Statistical analyses were performed using unpaired two-tailed t -test. ns, p > 0.05. Source data are provided as a Source Data file.

Article Snippet: Wells were subsequently washed with wash buffer and were incubated with biotinylated Rabbit Anti-Human VEGF detection antibody (1:800 in dilution buffer, #500-P10BT, Peprotech) for 2 h at RT.

Techniques: Expressing, Western Blot, Derivative Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Selective targeting of ligand-dependent and -independent signaling by GPCR conformation-specific anti-US28 intrabodies

doi: 10.1038/s41467-021-24574-y

Figure Lengend Snippet: a U251 with inducible US28 expression (No Nb) were transduced with lentiviral vector expressing inducible non-US28 targeting intrabody-mVenus (Irr Nb mV) or VUN103-mVenus (VUN103 mV). Cells were (induced) or were not (Non-induced) induced with doxycycline. US28 was detected using an anti-US28 antibody (US28, red). Nanobody binding was detected using the mVenus-tag (Nb mV, green). Representative data of three independent experiments. b Cells were seeded in hanging droplet plates and expression of US28 (and intrabodies) were or were not induced with doxycycline. Representative data of three independent experiments. c Spheroid areas were quantified and normalized to the spheroid area of non-induced US28 negative (US28 neg) spheroid ( n = 6 spheroids for non-induced and induced No Nb samples; n = 8 spheroids for non-induced Irr Nb mV samples; n = 12 spheroids for induced Irr Nb mV samples; n = 7 spheroids for non-induced VUN103 mV samples; n = 11 spheroids for induced VUN103 mV samples). d Western blot of phospho-STAT3 (p-STAT3), total STAT3, immediate early proteins (IE), intrabody-mVenus (mVenus) and actin of U251 cells (No Nb), U251cells transduced with a lentiviral vector expressing inducible non-US28 targeting intrabody-mVenus (Irr Nb mV) or VUN103-mVenus (VUN103 mV). Cells were not infected or infected with HCMV wild-type virus (WT). Representative blot shown from three independent experiments using independent biological replicates. Samples are derived from the same experiment and blots were processed in parallel. e U251 cells (No Nb), U251cells transduced with a lentiviral vector expressing inducible non-US28 targeting intrabody-mVenus (Irr Nb mV) or VUN103-mVenus (VUN103 mV)were uninfected or infected with HCMV wild-type virus (WT) or HCMV virus lacking US28 (HCMV ΔUS28). Conditioned serum of infected cells was harvested and VEGF levels were determined using ELISA ( n = 3 independent experiments). All data are plotted as mean ± SD. Statistical analyses were performed using an unpaired two-tailed t -test. ns, p > 0.05. Source data are provided as a Source Data file.

Article Snippet: Wells were subsequently washed with wash buffer and were incubated with biotinylated Rabbit Anti-Human VEGF detection antibody (1:800 in dilution buffer, #500-P10BT, Peprotech) for 2 h at RT.

Techniques: Expressing, Transduction, Plasmid Preparation, Binding Assay, Western Blot, Infection, Virus, Derivative Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test