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: The Journal of Neuroscience

Article Title: CD44 Signaling Mediates High Molecular Weight Hyaluronan-Induced Antihyperalgesia

doi: 10.1523/JNEUROSCI.2695-17.2017

Figure Lengend Snippet: Role of peptidergic (IB4−) and nonpeptidergic (IB4+) nociceptors in LMWH-induced hyperalgesia. A, Separate groups of rats received a single intrathecal injection of one of two neurotoxins, isolectin B4-saporin (IB4sap, selective for nonpeptidergic neurons, 3.2 μg, light gray bar), or [Sar9,Met(O2)11] substance P-saporin (SSPsap, selective for SP-containing fibers, 100 ng, dark gray bar), or their combination (black bar). A control group (white bar) received saline. LMWH (1 μg) was injected intradermally on the dorsum of the hindpaw 14 d later. Significant attenuation of LMWH-induced hyperalgesia was observed in all neurotoxin-treated groups (IB4sap: t(22) = 4.162, ***p = 0.0002; SSPsap: t(22) = 5.341, ****p < 0.0001; combination: t(22) = 7.762, ****p < 0.0001, compared with the control group; unpaired Student's t test). B, One week later, the control group (white bar) and the group treated with the combination of toxins (black bar), shown in A, were each further divided into 2 groups, which received an intradermal injection of either 8-bromo cAMP or ψεRACK (both 1 μg), on the dorsum of the hindpaw. At that time point, the mechanical nociceptive thresholds were not significantly different from pre-LMWH baseline (8-bromo cAMP: control group: t(5) = 0.9715, p = 0.1880; combination group: t(5) = 1.321, p = 0.1219; ψεRACK: control group: t(5) = 1.305, p = 0.1244; combination group: t(5) = 0.6697, p = 0.2664, all nonsignificant, when the mechanical nociceptive threshold before LMWH injection and immediately before 8-bromo cAMP or ψεRACK injection are compared, paired Student's t test). Robust mechanical hyperalgesia was observed in all groups, 30 min after injection (8-bromo cAMP, control: t(5) = 21.02, p < 0.0001; combination: t(5) = 4.776, p = 0.0025; ψεRACK, control: t(5) = 31.30, p < 0.0001; combination: t(5) = 6.173, p = 0.0008, when the mechanical nociceptive thresholds were compared with baseline thresholds; paired Student's t test), although in the groups pretreated with the combination of the two toxins, there was attenuation compared with the control groups (8-bromo cAMP-treated groups: t(10) = 3.907, *p = 0.0015; ψεRACK-treated groups: t(10) = 3.501, **p = 0.0029; unpaired Student's t test). A, n = 12 paws per group. B, n = 6 paws per group.

Article Snippet: Both IB4-saporin and SSP-saporin (from Advanced Targeting Systems) were diluted in saline to doses previously shown to deplete nonpeptidergic (3.2 μg/rat for IB4-saporin) and peptidergic (100 ng/rat for SSP-saporin) fibers ( Vulchanova et al., 2001 ; Wiley et al., 2007 ; Choi et al., 2012 ).

Techniques: Injection

Journal: Neuron

Article Title: Distinct modes of presynaptic inhibition of cutaneous afferents and their functions in behavior

doi: 10.1016/j.neuron.2019.02.002

Figure Lengend Snippet: A. Recording configuration denoting light stimulation of the dorsal root entry zone. B. CCKiCre-labeled interneuron induced DRPs in neonatal or juvenile mice are completely or partially blocked by GABAAR antagonists, respectively. Shaded region demarcates area under the DRP calculated in C. C. Composite data, integrating the area under the filtered DRP up to its peak in both neonatal and juvenile mice. Asterisks denote statistical significance from no effect. D. Transient VGlut3+ interneuron (labeled using VGlut3Cre, Lbx1Flpo mice) activated DRPs depend on ionotropic glutamate receptors, particularly NMDARs, and are enhanced by GABAAR antagonists. E. Composite data, integrating area under the full filtered DRP for each antagonist. F. Application of tetrodotoxin (TTX, 1uM) greatly diminishes the transient VGlut3+ interneuron optogenetically evoked DRP, which is partially recovered by the addition of 600 μM 4-AP. Electrically evoked DRPs were completely blocked by TTX and could not be recovered with the addition of 4-AP (400μM-1mM). G. Composite data of the effect of TTX (1 μM) and 4-AP (400–600μM) on optogenetically evoked DRPs. One-way ANOVA, F(1.241,2.482)=307.2, p=.0011. Asterisks denote Tukey’s multiple comparison post hoc test. Physiology scale bars denote 0.05mV, 100ms. H. Immunohistochemistry of transient VGlut3+ interneurons and CCKiCre labeled interneurons. Upper: Sagittal sections from CCKiCre; Rosa26LSL-ChR2 mouse counterstained for IB4 and VGlut1; Lower: Sagittal sections from VGlut3Cre; Lbx1Flpo; Rosa26LSL-FSF-Synaptohpysin-GFP mouse counterstained for VGlut1 and IB4. Inset is high magnification of close proximity of transient VGlut3 interneuron terminals to VGlut1 presumptive afferent terminals. (*** p< .001, ** p<.01, * p<.05).

Article Snippet: The following primary antibodies and concentrations were used: guinea pig anti-VGlut1 (Millipore AB5905, 1:1000, RRID:AB_2301751); mouse anti-NR1 (Millipore MAB 363, 1:500, RRID:AB_94946), chicken anti-GFP (Aves, GFP-1020, 1:1000, RRID:AB_10000240), IB4–647 (LifeTech {"type":"entrez-nucleotide","attrs":{"text":"I32450","term_id":"1823241","term_text":"I32450"}} I32450 , 1:500), rabbit anti-NF200 (Sigma N4142, 1:1000, RRID:AB_477272).

Techniques: Labeling, Immunohistochemistry

Journal: Molecular Pain

Article Title: Sigma-1 receptors and progesterone metabolizing enzymes in nociceptive sensory neurons of the female rat trigeminal ganglia: A neural substrate for the antinociceptive actions of progesterone

doi: 10.1177/17448069211069255

Figure Lengend Snippet: Antibodies used for western blotting and fluorescent immunohistochemistry.

Article Snippet: Isolectin IB 4 Biotin conjugate , Fisher Scientific , I21414 , Streptavidin 488 conjugate , Non-peptidergic neurons.

Techniques: Western Blot, Immunohistochemistry

Journal: Molecular Pain

Article Title: Sigma-1 receptors and progesterone metabolizing enzymes in nociceptive sensory neurons of the female rat trigeminal ganglia: A neural substrate for the antinociceptive actions of progesterone

doi: 10.1177/17448069211069255

Figure Lengend Snippet: Sigma-1 receptors are expressed in nociceptive and non-peptidergic neurons in the female rat trigeminal ganglia. Immunofluorescent staining of sigma-1 receptor (Sig-1R; red) (A), sodium channel 1.8 (Nav 1.8; nociceptors; blue) (B), and merged image (C) at 20X magnification. Sig-1 R (red) (D), Nav 1.8 (blue) (E), and merged image (F) at 40X magnification. Immunofluorescent staining of Sig-1R (red) (H), isolectin IB4 (non-peptidergic neurons; green) (G), and merged image (I) at 20X magnification. Sig-1 R (red) (K), isolectin IB4 (J), and merge images (L) at 40X magnification. Arrows indicate cells with protein coexpression.

Article Snippet: Isolectin IB 4 Biotin conjugate , Fisher Scientific , I21414 , Streptavidin 488 conjugate , Non-peptidergic neurons.

Techniques: Staining

Journal: Molecular Pain

Article Title: Sigma-1 receptors and progesterone metabolizing enzymes in nociceptive sensory neurons of the female rat trigeminal ganglia: A neural substrate for the antinociceptive actions of progesterone

doi: 10.1177/17448069211069255

Figure Lengend Snippet: Sigma-1 receptors and progesterone metabolizing enzymes, 5α-reductase and 3α-hydroxysteroid dehydrogenase, are highly expressed across various neuron populations in female rat trigeminal ganglia neurons. Quantification of the percentage of Sigma-1 receptors (Sig-1R), 5α-reductase, or 3α-hydroxysteroid dehydrogenase (3α-HSD) that are co-localized within the following neuron populations: protein gene product 9.5 (PGP9.5; neurons and nerve fibers; A), neurofilament heavy (NF200; myelinated neurons; B), sodium channel 1.8 (Nav1.8; nociceptors; C), isolectin IB4 (IB4, non-peptidergic neurons; D), transient receptor potential vanilloid 1 (TRPV1; nociceptor subpopulation; E), or calcitonin gene-related peptide (CGRP; peptidergic neurons; F).

Article Snippet: Isolectin IB 4 Biotin conjugate , Fisher Scientific , I21414 , Streptavidin 488 conjugate , Non-peptidergic neurons.

Techniques:

Journal: Molecular Pain

Article Title: Sigma-1 receptors and progesterone metabolizing enzymes in nociceptive sensory neurons of the female rat trigeminal ganglia: A neural substrate for the antinociceptive actions of progesterone

doi: 10.1177/17448069211069255

Figure Lengend Snippet: 5α-reductase expression in non-peptidergic nociceptive sensory neurons within the trigeminal ganglia of female rats. Immunofluorescent staining of sodium channel 1.8 (Nav 1.8; nociceptor; green) (A), 5α-reductase (blue) (B), and merged image (C) at 20X magnification. Nav 1.8 (green) (D), 5α-reductase (blue) (E), and merged images (F) at 40X magnification. Immunofluorescent staining of isolectin IB4 (non-peptidergic neurons; green) (G), 5α-reductase (blue) (H), and merged image (I) at 20X magnification. Isolectin IB4 (green) (J), 5α-reductase (blue) (K), and merged image (L) at 40X magnification. Arrows indicate cells with overlapping protein and enzyme immunoreactivity.

Article Snippet: Isolectin IB 4 Biotin conjugate , Fisher Scientific , I21414 , Streptavidin 488 conjugate , Non-peptidergic neurons.

Techniques: Expressing, Staining

Journal: Molecular Pain

Article Title: Sigma-1 receptors and progesterone metabolizing enzymes in nociceptive sensory neurons of the female rat trigeminal ganglia: A neural substrate for the antinociceptive actions of progesterone

doi: 10.1177/17448069211069255

Figure Lengend Snippet: 3α-hydroxysteroid dehydrogenase expression in non-peptidergic nociceptive sensory neurons within the trigeminal ganglia of female rats. Immunofluorescent staining of sodium channel 1.8 (Nav 1.8; nociceptor; blue) (B), 3α-hydroxysteroid dehydrogenase (3α-HSD; red) (A), and merged image (C) at 20X magnification. Nav 1.8 (blue) (E), 3α-HSD (red) (D), and merged images (F) at 40X magnification. Immunofluorescent staining of isolectin IB4 (non-peptidergic neurons; green) (G), 3α-HSD (blue) (H), and merged image (I) at 20X magnification. Isolectin IB4 (J), 3α-HSD (blue) (K), and merged image (L) at 40X magnification. Arrows indicate cells with overlapping protein and enzyme immunoreactivity.

Article Snippet: Isolectin IB 4 Biotin conjugate , Fisher Scientific , I21414 , Streptavidin 488 conjugate , Non-peptidergic neurons.

Techniques: Expressing, Staining

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