Journal: FEBS letters

Article Title: Hypoxia inducible factor-1α suppresses Peroxiredoxin 3 expression to promote proliferation of CCRCC cells.

doi: 10.1016/j.febslet.2014.07.030

Figure Lengend Snippet: Fig. 2. The expression of Prx3 is regulated by VHL. (A–C) The mRNA and protein levels of the indicated genes were respectively detected by real-time quantitative RT-PCR and Western blot. RCC4/VHL and Caki-1 cells were infected with retroviral vectors harboring shRNAs against VHL (shVHL) or scrambled negative control (NC) (B and C). EV represents empty vector. The column represents mean with bar as S.D. of three independent experiments with triplicate samples (⁄P < 0.05; ⁄⁄P < 0.01).

Article Snippet: The proteins were probed by antibodies against human Prx3 (sc59661, Santa Cruz Biotech), HIF-1a (610958, BD Transduction Laboratories), VHL (NB100-1899, Novus biologicals), PDK1 (KAPPK112, Enzo life sciences) and Actin (JLA20, Merck).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Infection, Retroviral, Negative Control, Plasmid Preparation

Journal: FEBS letters

Article Title: Hypoxia inducible factor-1α suppresses Peroxiredoxin 3 expression to promote proliferation of CCRCC cells.

doi: 10.1016/j.febslet.2014.07.030

Figure Lengend Snippet: Fig. 3. Prx3 is negatively regulated by HIF-1a. The mRNA and protein levels of the indicated genes were respectively detected by real-time quantitative RT-PCR and Western blot. (A) RCC4/VHL cells were exposed to normal air or hypoxia (1% O2) for 12 h. (B) RCC4 cells were stably transfected with shRNAs against HIF-1a (a1and a2). (C) Caki-1 cells transfected with shRNAs against HIF-1a (a1 and a2) were incubated in normoxia or hypoxia for 12 h. Columns, means of three determinations; bars, S.D. (⁄P < 0.05; ⁄⁄P < 0.01).

Article Snippet: The proteins were probed by antibodies against human Prx3 (sc59661, Santa Cruz Biotech), HIF-1a (610958, BD Transduction Laboratories), VHL (NB100-1899, Novus biologicals), PDK1 (KAPPK112, Enzo life sciences) and Actin (JLA20, Merck).

Techniques: Quantitative RT-PCR, Western Blot, Stable Transfection, Transfection, Incubation

Journal: FEBS letters

Article Title: Hypoxia inducible factor-1α suppresses Peroxiredoxin 3 expression to promote proliferation of CCRCC cells.

doi: 10.1016/j.febslet.2014.07.030

Figure Lengend Snippet: Fig. 4. Two HREs in promoter of PRDX3 are essential for HIF-1a transactivity. (A) 293T cells were transfected with luciferase reporter plasmids driven by four putative HREs in PRDX3 promoter, which are shown on the top and middle panels, and were grown in the normal air or hypoxia for 12 h. In top diagram of putative HREs (black ovals), empty circles represent the transcriptional start point of PRDX3. (B) 293T cells were transfected with luciferase reporter plasmids driven by four putative HREs in PRDX3 promoter together with the indicated doses of HIF-1a and grown in the normal air for 36 h. Protein levels of HIF-1a were detected by western blot with Actin as a loading control (low panel). (C) Luciferase reporter plasmids driven by the HREs or CG/AA mutated HRE sequences as indicated were transfected together with HIF-1a expressing vector or empty vector into 293T cells for 36 h in normal air. All the relative luciferase activities of PRDX3 promoter were normalized by pSV40-Renilla and estimated as the relative folds against cells under normal air (A, lower panel) or empty vector-transfected cells (B and C). (D) RCC4/VHL cells were grown under normoxia and hypoxia for 12 h. Chromatin immunoprecipitation assay was performed as described in Section 2. The column represents mean with bar as S.D. of three independent experiments with triplicate samples (⁄P < 0.05; ⁄⁄P < 0.01).

Article Snippet: The proteins were probed by antibodies against human Prx3 (sc59661, Santa Cruz Biotech), HIF-1a (610958, BD Transduction Laboratories), VHL (NB100-1899, Novus biologicals), PDK1 (KAPPK112, Enzo life sciences) and Actin (JLA20, Merck).

Techniques: Transfection, Luciferase, Western Blot, Control, Expressing, Plasmid Preparation, Chromatin Immunoprecipitation

Journal: Applied and Environmental Microbiology

Article Title: Bovine Norovirus: Carbohydrate Ligand, Environmental Contamination, and Potential Cross-Species Transmission via Oysters ▿ †

doi: 10.1128/AEM.00671-10

Figure Lengend Snippet: Flow cytometric analysis of the binding of BEC28 VLPs and the GS1-B4 isolectin to rat Ggta1-transfected or control mock-transfected human HEK293 cells. The respective negative controls correspond to either cells incubated in the absence of the lectin or to cells incubated in the presence of the VLPs followed by incubation with an irrelevant rabbit antiserum and FITC-labeled anti-rabbit IgG. The log of fluorescence intensities in arbitrary units is plotted against the cell number.

Article Snippet: The lectin from Griffonia simplicifolia B4 isolectin 1 (GS1-B4), either peroxidase or fluorescein isothiocyanate (FITC) conjugated, which recognizes α1,3-linked terminal galactosyl residues, was purchased from Vector Laboratories (Burlingame, CA) and from EY Laboratories (San Mateo, CA), respectively.

Techniques: Binding Assay, Transfection, Control, Incubation, Labeling, Fluorescence

Journal: The Journal of Neuroscience

Article Title: Mediation of Movement-Induced Breakthrough Cancer Pain by IB4-Binding Nociceptors in Rats

doi: 10.1523/JNEUROSCI.1212-16.2017

Figure Lengend Snippet: Tactile hypersensitivity and movement-induced BTP is dependent on IB4 positive fibers. A, Spinal administration of IB4-SAP attenuated tumor-induced tactile hypersensitivity. Vehicle-treated tumor-bearing rats demonstrated tactile hypersensitivity with paw withdrawal thresholds significantly lower than precancer implantation baselines. ***p < 0.01 versus BL. Ablation of IB4-binding fibers by spinal administration of IB4-SAP attenuated tumor-induced tactile hypersensitivity, with paw withdrawal thresholds significantly higher compared with SAP control rats. #p < 0.05 versus SAP. Spinal ablation of TRPV1-expressing terminals in the spinal dorsal horn by spinal administration of capsaicin failed to eliminate tumor-induced tactile hypersensitivity, with paw withdrawal thresholds significantly lower than precancer baselines. ***p < 0.01 versus BL. B, Group comparison of difference scores demonstrates that movement induced CPA in vehicle-treated rats compared with sham controls. *p < 0.05 versus sham. Ablation of IB4-binding fibers blocked movement-induced CPA. In contrast, ablation of TRPV1-expressing terminals in the spinal dorsal horn failed to block movement-induced CPA. **p < 0.01 versus shams. Graphs are mean ± SEM. Sham, n = 10 capsaicin, n = 10 IB4-SAP; cancer, n = 9 SAP, n = 8 capsaicin vehicle, n = 11 IB4-SAP, and n = 10 capsaicin.

Article Snippet: To determine the effect of eliminating input from IB4-binding fibers, separate groups of rats received spinal administration of IB4-Saporin (IB4-SAP, 3.2 μg/20 μl saline; Advanced Targeting Systems) or the control, blank-SAP (3.2 μg/20 μl saline; Advanced Targeting Systems) followed by a 10 μl flush of saline.

Techniques: Binding Assay, Expressing, Blocking Assay

Journal: The Journal of Biological Chemistry

Article Title: Comprehensive characterization of complex glycosphingolipids in human pancreatic cancer tissues

doi: 10.1016/j.jbc.2023.102923

Figure Lengend Snippet: Binding of antibodies and lectins to subfractions of neutral glycosphingolipids obtained from pooled human pancreatic ductal adenocarcinoma tissues and surrounding normal tissues. Thin-layer chromatogram with anisaldehyde detection ( A ) and autoradiograms obtained by binding Galα4Gal-recognizing P-fimbriated Escherichia coli strain 291-15 ( B ), Galβ4GlcNAc-recognizing Erythrina crista galli lectin ( C ), monoclonal antibodies directed against the blood group Le a ( D ), Le b ( E ), and A ( F ) determinants, and terminal Galα-recognizing Griffonia simplicifolia IB4 lectin ( G ). The separation of glycosphingolipids and subsequent chromatogram-binding assays were performed as described in “ ”. Lanes: lane 1, neutral glycosphingolipid fraction of pooled normal pancreatic tissue (NT), 40 μg; lane 2, neutral glycosphingolipid fraction of pooled pancreatic ductal adenocarcinoma tissue (T), 40 μg; lane 3, reference Le b hexosylceramide (Le b -6, Fucα2Galβ3(Fucα4)GlcNAcβ3Galβ4Glcβ1Cer), 4 μg; lane 4, reference A type 2 hexosylceramide (A6-2, GalNAcα3(Fucα2)Galβ4GlcNAcβ3Galβ4Glcβ1Cer), 4 μg; lane 5, reference B type 2 hexosylceramide (B6-2, Galα3(Fucα2)Galβ4GlcNAcβ3Galβ4Glcβ1Cer), 4 μg. The Roman numbers to the left of chart A denote the number of carbohydrate units in the bands.

Article Snippet: Griffonia simplicifolia lectin IB4 , − , Advanced Targeting System , 1:200 , − , Galα.

Techniques: Binding Assay, Bioprocessing

Journal: The Journal of Biological Chemistry

Article Title: Comprehensive characterization of complex glycosphingolipids in human pancreatic cancer tissues

doi: 10.1016/j.jbc.2023.102923

Figure Lengend Snippet: Carbohydrate-binding ligands used in chromatogram-binding assays

Article Snippet: Griffonia simplicifolia lectin IB4 , − , Advanced Targeting System , 1:200 , − , Galα.

Techniques: Binding Assay, Plasmid Preparation

Journal: Asian-Australasian Journal of Animal Sciences

Article Title: Alpha-1,3-galactosyltransferase-deficient miniature pigs produced by serial cloning using neonatal skin fibroblasts with loss of heterozygosity

doi: 10.5713/ajas.16.0010

Figure Lengend Snippet: Flow cytometric analysis of fibroblasts from α GT-deficient piglets. This histogram plots a single parameter (FITC intensity, horizontal axis) against the number of cells detected (vertical axis). Thick solid line, wild type cells; Thick dotted line, heterozygous α GT KO cells; Thin dotted line, homozygous α GT KO cells; Thin solid line, unstained cells. α GT, alpha-1,3-galactosyltransferase; KO, knockout; FITC, fluorescein isothiocyanate.

Article Snippet: Then cells were labeled with fluorescein isothiocyanate (FITC)-conjugated IB4 and analyzed using BD FACSCalibur Cell Analyzer (BD Biosciences, San Jose, CA, USA).

Techniques: Knock-Out