Journal: Journal of molecular biology

Article Title: Cullin E3 ligase activity is required for myoblast differentiation

doi: 10.1016/j.jmb.2017.02.012

Figure Lengend Snippet: A & B) Immunofluorescence analysis of C2C12 cells differentiated for 5 days in presence or absence of 330nM MLN4924, stained with antibodies against sarcomeric myosin heavy chain (MyHC) and desmin (A), or alpha-actinin (B). DAPI was used as counterstain in A and B. Scale bar = 50μm. C) Immunoblot analysis of pan-sarcomeric myosin heavy chain (MyHC) and myogenin expression in whole cell lysates of C2C12 cells either in proliferation (Pro), or after 5 days in differentiation (Diff) medium with 300nM MLN4924 (+) or vehicle control (DMSO, −). GAPDH was used as loading control. D–F) Relative normalized protein levels of nedd8 protein levels (80kDa band, D), myogenin (E) and pan-sarcomeric myosin heavy chain (MyHC, F) in MLN4924 (MLN) treated C2C12 cells five days after differentiation compared to controls (CTL). Three independent samples were run per group; p-values are indicated in the figure.

Article Snippet: Singer), nedd8 (Cell Signaling; Danvers, MA), pan-sarcomeric myosin heavy chain (clone 1025, DSHB), myogenin (clone F5D, from DSHB and Abcam), ZBTB38 (R&D Systems; Minneapolis, MN), Bhlhe41 ( {"type":"entrez-protein","attrs":{"text":"ARP33575","term_id":"1190164483","term_text":"ARP33575"}} ARP33575 ; AVIVA Systems Biology, San Diego, CA), Id1 (sc-133104; Santa Cruz Biotechnologies), or GAPDH (Santa Cruz Biotechnologies) in blocking solution overnight at 4°C.

Techniques: Immunofluorescence, Staining, Western Blot, Expressing

Journal: Journal of molecular biology

Article Title: Cullin E3 ligase activity is required for myoblast differentiation

doi: 10.1016/j.jmb.2017.02.012

Figure Lengend Snippet: A) Immunofluorescence analysis of primary cultures of mouse muscle stem cells after 5 days of differentiation. Cells were differentiated in presence of 330nM MLN4924 or vehicle control (DMSO), and stained with antibodies against desmin and pan-sarcomeric myosin heavy chain (MyHC). DAPI was used as counterstain. Scale bar = 100μm. B) Immunoblot analysis of cullin-1 and pan-sarcomeric myosin heavy chain (MyHC) protein levels in total lysates of differentiated primary mouse muscle stem cell cultures, treated with 330nM MLN4924 (+) or vehicle control (DMSO, −). GAPDH and ponceau stained actin band were used as loading controls. C & D) Immunofluorescence analysis of human muscle stem cells after 5 days of differentiation in presence of 330nM MLN4924 or vehicle control (DMSO). Cells were stained with antibodies against slow myosin heavy chain 7 (MyHC; C) or myogenin (D). DAPI and fluorescently labeled WGA were used as counterstains. Scale bar = 100μm.

Article Snippet: Singer), nedd8 (Cell Signaling; Danvers, MA), pan-sarcomeric myosin heavy chain (clone 1025, DSHB), myogenin (clone F5D, from DSHB and Abcam), ZBTB38 (R&D Systems; Minneapolis, MN), Bhlhe41 ( {"type":"entrez-protein","attrs":{"text":"ARP33575","term_id":"1190164483","term_text":"ARP33575"}} ARP33575 ; AVIVA Systems Biology, San Diego, CA), Id1 (sc-133104; Santa Cruz Biotechnologies), or GAPDH (Santa Cruz Biotechnologies) in blocking solution overnight at 4°C.

Techniques: Immunofluorescence, Staining, Western Blot, Labeling

Journal: Journal of molecular biology

Article Title: Cullin E3 ligase activity is required for myoblast differentiation

doi: 10.1016/j.jmb.2017.02.012

Figure Lengend Snippet: A) Immunofluorescence of C2C12 cells five days after differentiation in presence of 330nM MLN4924 alone, in combination with 10μM 5-aza-dC (Aza), or vehicle control (DMSO). Cells were stained with an antibody against alpha-actinin (green in overlay). DAPI (blue in overlay) was used as counterstain. Scale bar = 100μm. B) Fraction analysis of nuclei per alpha-actinin positive cells in 330nM MLN4924 (MLN) treated, or 330nM MLN4924 and 10μM Aza treated C2C12 five days after differentiation, compared to vehicle control (CTL/DMSO). Sample size (total counted nuclei) and p-values are indicated in the figure. C) Analysis of alpha-actinin positive cells per mm2 in presence of MLN4924 (MLN) alone, in combination with 10μM Aza, or in control (CTL). Sample size of alpha actinin positive cells, and p-values are indicated in the figure. D) Quantitative PCR analysis of relative normalized (against GAPDH) myogenin expression in proliferative C2C12 myoblasts cultures (Pro), compared to C2C12 differentiated for five days in presence of 330nM MLN4924 (MLN) alone, or in combination with 10μM Aza, or vehicle treated control (CTL, DMSO). Sample size (biological replicates) and p-values are indicated in the figure. (n.s. = non-significant). E) Immunoblot analysis of sarcomeric myosin heavy chain (MyHC) and nedd8 protein levels in differentiated C2C12 cells after five days in culture. Samples are total protein lysates of cells treated with 330nM MLN4924 (MLN) alone, or in combination with 10μM Aza. F, G) Fusion index (F) and cluster analysis of nuclei/myotube (G) of 330nM MLN4924 (MLN) treated, or 330nM MLN4924 and 10μM Aza treated C2C12 five days after differentiation, compared to vehicle control (CTL/DMSO). Sample size and p-values (in F) are indicated in the figure.

Article Snippet: Singer), nedd8 (Cell Signaling; Danvers, MA), pan-sarcomeric myosin heavy chain (clone 1025, DSHB), myogenin (clone F5D, from DSHB and Abcam), ZBTB38 (R&D Systems; Minneapolis, MN), Bhlhe41 ( {"type":"entrez-protein","attrs":{"text":"ARP33575","term_id":"1190164483","term_text":"ARP33575"}} ARP33575 ; AVIVA Systems Biology, San Diego, CA), Id1 (sc-133104; Santa Cruz Biotechnologies), or GAPDH (Santa Cruz Biotechnologies) in blocking solution overnight at 4°C.

Techniques: Immunofluorescence, Staining, Real-time Polymerase Chain Reaction, Expressing, Western Blot

Journal: Brain : a journal of neurology

Article Title: Connexin43 mimetic peptide reduces vascular leak and retinal ganglion cell death following retinal ischaemia.

doi: 10.1093/brain/awr338

Figure Lengend Snippet: Figure 3 Three hours of hypoxia and 6 h of reperfusion lead to significant endothelial cell death in vitro in rat brain microvascular endothelial cells (RBMVEC; A). Non-specific gap junction blocker carbenoxolone, non-specific hemichannel blocker LaCl3, and connexin43 (Cx43) mimetic peptide protected endothelial cells against hypoxic injury, with the number of viable cells significantly higher than no treatment, while scrambled peptide did not have any protective effects. The number of viable cells was expressed as percentage of the control without hypoxia. Hypoxia and reperfusion also lead to significant propidium iodide dye uptake into primary rat brain microvascular endothelial cells indicating open hemichannels (B). Carbenoxolone, LaCl3 and connexin43 mimetic peptide significantly prevented dye uptake compared to no treatment, indicating hemichannel closure, while scrambled peptide did not have any effect. Stars denote statistical significance when compared to the control group or compared between groups in brackets; P 5 0.05.

Article Snippet: Rat brain microvascular endothelial cells (R840K-05a, Cell Applications) were plated into 24-well plates (1 105 cells/well) in rat brain microvascular endothelial cell growth media (R819K-500, Cell Applications) and allowed to settle for 16 h. Medium was then removed and replaced with Dulbecco’s Modified Eagle’s Medium/F12 containing 0.5% foetal bovine serum and 1% glutamine.

Techniques: In Vitro, Control

Journal: Journal of molecular biology

Article Title: Feline Leukemia Virus-B Envelope Together With its GlycoGag and Human Immunodeficiency Virus-1 Nef Mediate Resistance to Feline SERINC5.

doi: 10.1016/j.jmb.2021.167421

Figure Lengend Snippet: Figure 5. Effect of SER5 on HIV-1 particles pseudotyped with different envelopes (A) HIV-1 reporter vectors (3-plasmid system, no Nef) pseudotyped with envelopes of HIV-1 BaL (red), FeLV-A (green), FeLV-B (blue) or amphotropic (ampho) Env (orange) were produced in the presence of increasing amounts of feline SERINC5 (feSER5) or (B) human SERINC5 (huSER5) (0, 100, 200 or 400 ng). Viral infectivity was determined after normalization for reverse transcriptase activity by quantification of luciferase activity. Shown are means plus standard deviations (error bars) of a representative experiment performed three times in triplicate. Asterisks represent statistically significant differences: P value <0.001 extremely significant (***), 0.001 to 0.01 very significant (**), 0.01 to 0.05 significant (*), >0.05 not significant (ns). (C) SERINC5 stability in the presence of increasing amounts of FeLV-B envelope. HEK293T cells were transfected with fe/huSER5 and increasing amounts of FeLV-B envelope expression plasmids or empty vector as control. Cells were harvested and analyzed by immunoblotting using anti-p85/70, anti-HA and anti-tubulin antibodies. a p85/70 detects envelope (SU-TM p85) and SU only (p70), a HA detects HA-SER5.

Article Snippet: The glycoGag expression in the FeLV plasmids was evaluated by using anti-FeLV p27 antibody (1:7500 dilution; PF12J-10A: sc-65623; Santa Cruz Biotechnology, Inc. Bergheimer Heidelberg.

Techniques: Plasmid Preparation, Produced, Infection, Reverse Transcription, Activity Assay, Luciferase, Transfection, Expressing, Control, Western Blot

Journal: Journal of molecular biology

Article Title: Feline Leukemia Virus-B Envelope Together With its GlycoGag and Human Immunodeficiency Virus-1 Nef Mediate Resistance to Feline SERINC5.

doi: 10.1016/j.jmb.2021.167421

Figure Lengend Snippet: Figure 6. Generation of FeLV glycoGag expression constructs. (A) Sequences of FeLV-A and FeLV-B glycoGags. The highlighted amino acids correspond to disagreements, which are colored according to their side chain chemistry. Cytoplasmic and transmembrane domains are indicated. (B) Plasmids expressing the HA-tagged FeLV glycoGag were produced by amplifying the glycoGag from pFGA-5 and pFGB (FeLV-A and FeLV-B glycoGag respectively), yielding a 9.6 kDa protein. (C) HEK293T cells were transfected with the generated expression plasmids, harvested two days post-transfection and analyzed by immunoblotting using anti-HA and anti-tubulin antibodies.

Article Snippet: The glycoGag expression in the FeLV plasmids was evaluated by using anti-FeLV p27 antibody (1:7500 dilution; PF12J-10A: sc-65623; Santa Cruz Biotechnology, Inc. Bergheimer Heidelberg.

Techniques: Expressing, Construct, Produced, Transfection, Generated, Western Blot

Journal: Journal of molecular biology

Article Title: Feline Leukemia Virus-B Envelope Together With its GlycoGag and Human Immunodeficiency Virus-1 Nef Mediate Resistance to Feline SERINC5.

doi: 10.1016/j.jmb.2021.167421

Figure Lengend Snippet: Figure 7. Effect of FeLV glycoGag on HIV-1 in the presence of SER5. (A) HIV-1 reporter vectors (3-plasmid system, no Nef) pseudotyped with HIV-1 BaL envelope were produced in the absence or presence of feline SERINC5 (feSER5) or human SERINC5 (huSER5) together with FeLV-A glycoGag, FeLV-B glycoGag or empty vector. Particles were normalized by RT activity and used for infections. The infectivity determined by quantification of luciferase activity. Shown are means plus standard deviations (error bars) of a representative experiment performed three times in triplicate. Asterisks represent statistically significant differences: P value <0.001 extremely significant (***), 0.001–0.01 very significant (**), 0.01–0.05 significant (*), >0.05 not significant (ns). (B) Effect of SERINC and glycoGag on HIV-1 cell-to-cell transmission. HIV-1 reporter vectors (3-plasmid system, no Nef) pseudotyped with HIV-1 envelope BaL were produced in the absence or presence of feline SERINC5 (feSER5) or human SERINC5 (huSER5) and FeLV-A or -B glycoGag. After transfection, virus producer HEK293T cells were co-cultured with HEK293T transfected with expression plasmids for human CD4 and human CCR5. The infectivity determined by quantification of luciferase activity of a vector that is detectable only after reverse transcription of the spliced luciferase gene. Shown are means plus standard deviations (error bars) of a representative experiment performed three times in triplicate. Asterisks represent statistically significant differences: P value <0.001 extremely significant (***), 0.001– 0.01 very significant (**), 0.01–0.05 significant (*), >0.05 not significant (ns).

Article Snippet: The glycoGag expression in the FeLV plasmids was evaluated by using anti-FeLV p27 antibody (1:7500 dilution; PF12J-10A: sc-65623; Santa Cruz Biotechnology, Inc. Bergheimer Heidelberg.

Techniques: Plasmid Preparation, Produced, Activity Assay, Infection, Luciferase, Transmission Assay, Transfection, Virus, Cell Culture, Expressing, Reverse Transcription

Journal: Journal of molecular biology

Article Title: Feline Leukemia Virus-B Envelope Together With its GlycoGag and Human Immunodeficiency Virus-1 Nef Mediate Resistance to Feline SERINC5.

doi: 10.1016/j.jmb.2021.167421

Figure Lengend Snippet: Figure 8. Effect of FeLV glycoGag or FeLV-B envelope on FIV in the presence of SER5. FIV particles (3- plasmid system) pseudotyped with FIV EE14 envelope were produced in the absence or presence of feline SERINC5 (feSER5) or human SERINC5 (huSER5) and FeLV glycoGag or in the absence of FIV EE14 envelope and glycoGag and pseudotyped by FeLV-B envelope. Particles were normalized by RT activity and used for infection. The infectivity determined by quantification of luciferase activity. Shown are means plus standard deviations (error bars) of a representative experiment performed three times in triplicate. Asterisks represent statistically significant differences: P value <0.001 extremely significant (***), 0.001–0.01 very significant (**), 0.01–0.05 significant (*), >0.05 not significant (ns).

Article Snippet: The glycoGag expression in the FeLV plasmids was evaluated by using anti-FeLV p27 antibody (1:7500 dilution; PF12J-10A: sc-65623; Santa Cruz Biotechnology, Inc. Bergheimer Heidelberg.

Techniques: Plasmid Preparation, Produced, Activity Assay, Infection, Luciferase

Journal: Journal of molecular biology

Article Title: Feline Leukemia Virus-B Envelope Together With its GlycoGag and Human Immunodeficiency Virus-1 Nef Mediate Resistance to Feline SERINC5.

doi: 10.1016/j.jmb.2021.167421

Figure Lengend Snippet: Figure 9. Comparison of delta Ct values of feSER5 in transfected HEK293T cells and in cat cells and surface expression of feSER5 in the presence of different FeLV proteins. (A) HEK293T cells were transfected with different amounts of feSER5 plasmid. The endogenous feSER5 were evaluated in feline CRFK cell lines and PBMC or macrophages from one cat. (B) HEK293T cells were cotransfected with feSER5-iHA and empty plasmid, Nef, MLV glycoGag , FeLV-A/B glycoGag or FeLV-A/B envelope. The percent of feSER5 positive cells was evaluated by flow cytometry analysis and the total of feSER5 positive cells were scaled to 100%. Shown are means plus standard deviations (error bars) of a representative experiment performed three times in triplicate. Asterisks represent statistically significant differences: P value <0.001 extremely significant (***), 0.001–0.01 very significant (**), 0.01– 0.05 significant (*), >0.05 not significant (ns). (C) Representative plots of one of three experiments showing the surface expression of feSER5-iHA (HA-Alexa Fluor 488). Green fluorescence was quantified using the FITC-A channel vs. FSC-A (10,000 cells were counted).

Article Snippet: The glycoGag expression in the FeLV plasmids was evaluated by using anti-FeLV p27 antibody (1:7500 dilution; PF12J-10A: sc-65623; Santa Cruz Biotechnology, Inc. Bergheimer Heidelberg.

Techniques: Comparison, Transfection, Expressing, Plasmid Preparation, Cytometry

Journal: Cancer research

Article Title: Promoting Thiol Expression Increases The Durability of Antitumor T cell Functions

doi: 10.1158/0008-5472.CAN-14-1084

Figure Lengend Snippet: (A) Activated human PBMCs cultured for seven days were restimulated overnight with anti-CD3 and stained for CD8, CD4, CD62L, and Annexin V for flow-cytometry-based analysis. (B) Human T cells were gated on CD8+CD44+CD62LhiCXCR3loCCR7hi (red) or CD8+CD44+CD62LloCXCR3hiCCR7lo (green) and analyzed for expression of: (Bi) c-SH using maleamide; p < 0.05, and (Bii) iGSH using monochlorobimane; p < 0.05. Data are representative of at-least seven experiments with similar results. (C) T cells were labeled with CFSE (1 μM) and stimulated or left unstimulated for 72 hours. Cells were then harvested and stained for CD8, Vβ13, CD62L and c-SH, and analyzed by flow cytometry. Cell cycle analysis was then performed using the unstained peak in FlowJo platform software for CD62L and c-SH on CD8+ cells. (N = 3) (D) PBMCs were cultured in IL-15 for 5 days and sorted as CD8+CD62Lhi or CD8+CD62Llo for RNA isolation. mRNA expression levels of antioxidant genes were determined by real-time PCR. All results are representative of three or more separate experiments. ** p <0.005; * p <0.05.

Article Snippet: Recombinant interleukin (IL)-15 and IL-2 were purchased from R & D Systems (Minneapolis, MN).

Techniques: Cell Culture, Staining, Flow Cytometry, Expressing, Labeling, Cell Cycle Assay, Software, Isolation, Real-time Polymerase Chain Reaction

Journal: Cancer research

Article Title: Promoting Thiol Expression Increases The Durability of Antitumor T cell Functions

doi: 10.1158/0008-5472.CAN-14-1084

Figure Lengend Snippet: Human tyrosinase epitope reactive TIL1383I TCR-transduced T cells were cultured in IL-2/IL-15 and used for further experimentation or sorting. (Ai) Cells were washed and labeled with fluorochrome-labeled antibodies for CD8, CD34 (TCR), CD62L and Mitotracker red. Cells were acquired on image stream (EMD Millipore Amnis) and analyzed using IDEAS v. 5.0 software. The cells were first gated on CD8+CD34+ and then on CD62Llo or CD62Lhi. The intensity of Mitotracker was compared in these cells. (Aii) In a parallel experiment, cells were acquired on BD LSR Fortessa and compared for Mitotracker staining and analyzed using FlowJo software. Experiment was repeated twice with similar results, p < 0.005. (B) Comparison between the mitochondrial DNA/nuclear DNA ratio (mDNA/nDNA) is shown, p < 0.05. (C) Real time PCR analysis for GLUT-1 expression between is shown, p < 0.005 (N = 3). (D) Glucose consumption assay was performed using 2NBGD and its florescence was compared between CD62Llo or CD62Lhi cells, p < 0.005 (N = 3). (E) RNA from sorted as CD62Llo or CD62Lhi cells and used to evaluate the differences between glycolysis pathway genes. Results shown were calculated from three separate experiments. ** p <0.005, * p <0.05.

Article Snippet: Recombinant interleukin (IL)-15 and IL-2 were purchased from R & D Systems (Minneapolis, MN).

Techniques: Cell Culture, Labeling, Software, Staining, Comparison, Real-time Polymerase Chain Reaction, Expressing

Journal: Cancer research

Article Title: Promoting Thiol Expression Increases The Durability of Antitumor T cell Functions

doi: 10.1158/0008-5472.CAN-14-1084

Figure Lengend Snippet: (A) TIL1383I TCR-transduced CD8+ preconditioned cells in rapamycin or L-NAC were analyzed for iGSH (p < 0.05; N = 3), and c-SH (p < 0.05; N = 3), as described in Methods. (B) TIL1383I TCR-transduced CD8+ pretreated with rapamycin or L-NAC was restimulated with either cognate peptide (human Tyrosinase) or control peptide (Mart-1) pulsed T2 cells. Cell death was determined using flow-cytometry-based Annexin V assay. Numbers represent MFI for Annexin V. (Ci) Human PBMCs were cultured in IL-15 for 3 days with rapamycin (250 nM), or L-NAC (5 mM) for 30 minutes, or kept untreated. Mitochondrial respiration was measured by determining oxygen consumption rate using a Seahorse analyzer as detailed in Methods. (Cii) Mean from three experiments with similar results is represented. (D) TIL1383I TCR-transduced CD8+ T cells preconditioned in rapamycin or L-NAC were analyzed for mitochondrial biogenesis genes and mitochondrial transcription factors using real time PCR analysis. (E) Human PBMCs treated with or without rapamycin or L-NAC were stained and analyzed using a fluorescent microscope (60X magnification). Five different fields were photographed for each slide for Mitotracker (red) and DAPI (blue) channels. ImageJ software was used to analyze the difference in Mitotracker intensity. Bar graph on the right represents an average from different experiments. Results are demonstrated as an average of two to three independent experiments with similar results. ** p <0.005, * p <0.05.

Article Snippet: Recombinant interleukin (IL)-15 and IL-2 were purchased from R & D Systems (Minneapolis, MN).

Techniques: Flow Cytometry, Annexin V Assay, Cell Culture, Real-time Polymerase Chain Reaction, Staining, Microscopy, Software

Journal: Cancer research

Article Title: Promoting Thiol Expression Increases The Durability of Antitumor T cell Functions

doi: 10.1158/0008-5472.CAN-14-1084

Figure Lengend Snippet: Human T cells transduced with TIL1383I TCR and cultured in IL-2/IL-15 were treated with/without rapamycin or L-NAC and analyzed for: (Ai) Glucose uptake using 2-NBDG on TCR-specific CD8+-gated viable cells as detailed in Methods. (Aii) Data represent MFI of 2NBDG from eight different samples. (B) ECAR levels using the Seahorse Analyzer, and (C) HIF-1α expression by Western blot. (Di) Persistence of TIL1383I CD8+ T cells in the spleen of NSG mice 24 h after adoptive transfer. Bar graph represents the number of cells recovered. Experiment was repeated twice. N=2. (Dii) Comparison of pre- and post-transfer recovered cells for CD62L expression. Experiment was repeated twice with 3-4 mice/group. (E) CD8+ transgenic T cells reactive to human gp100 were activated with cognate antigen in presence/absence of rapamycin along with IL-2 for three days. Equal number of cells were transferred into Rag−/− mice with subcutaneously established murine melanoma B16-F10. Rapamycin group received the maintenance dose of drug (i.p. dose=0.75mg/kg/day from 0-7 days). (Ei) Tumor growth was measured twice weekly as shown. Each group had 5 mice, and the experiment was repeated twice. **p <0.005, ****p <0.00005. Blood was obtained from each group of mice in Ei at the experimental end-point and donor CD8+Vβ13+ T cells were analyzed for: (Eii) CD62L expression, (Eiii) Percent of CD8+Vβ13+ cells (p < 0.05), and (Eiv) c-SH expression (p < 0.05).

Article Snippet: Recombinant interleukin (IL)-15 and IL-2 were purchased from R & D Systems (Minneapolis, MN).

Techniques: Transduction, Cell Culture, Expressing, Western Blot, Adoptive Transfer Assay, Comparison, Transgenic Assay

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