Journal: bioRxiv

Article Title: A dual role for peripheral GDNF signaling in nociception and cardiovascular reflexes

doi: 10.1101/627521

Figure Lengend Snippet: A . GDNF is increased in muscle after I/R compared to naïve animals (n=4 per group) as assessed by western blotting (WB). B . Representative image of GDNF distribution within myofibers in an injured forepaw muscle. DAPI was used to mark nuclei. Solid arrows: GDNF positive myofibers. Dashed arrows: GDNF negative myofiber. C . WB of GFRα1 in DRGs showed increased expression in the C7-T1 DRGs after I/R and in PenCON+I/R groups. Penα1 effectively prevented this upregulation (n=3 per group). D . Paw guarding is increased 1d after I/R and PenCON+I/R and this is partially prevented in Penα1+I/R animals. Mechanical withdrawal thresholds to von Frey hair (VFH) stimulation ( E ) and muscle squeezing ( F ) are decreased after I/R and PenCON+I/R. GFRα1 knockdown blocked these effects. G . Grip strength is decreased 1d after I/R and PenCON+I/R. This was partially reversed in Penα1+I/R animals. H . Only injured mice (I/R and PenCON+I/R) displayed a significant increase in MAP after forced exercise. The increase in MAP was not observed in Penα1+I/R mice. (D-H: n=12 per group. x p<0.5 vs Naïve, *p<0.5 vs Sham, **p<0.01 vs Penα1+I/R, ***p<0.001 vs Sham and Penα1+I/R, ****p<0.0001 vs Sham and Penα1+I/R, + p<0.01 vs Sham, ^ p<0.05 vs pre-exercise MAP; 1-way ANOVA with HSD post hoc test (A, C) or 2-way RM ANOVA with HSD post hoc test (D-H)).

Article Snippet: Embedded DRGs were stored at −80°C, sections (15 µm) were cut on a cryostat and mounted on slides and processed for GDNF receptor GFRα1 (goat anti-GFRα1, 1:100; cat no. AF-560, R&D Systems, Minneapolis, MN, USA) and P2X5 (rabbit anti-P2X5, 1:100; cat no. APR005 Alomone Labs, Jerusalem, Israel).

Techniques: Western Blot, Expressing

Journal: bioRxiv

Article Title: A dual role for peripheral GDNF signaling in nociception and cardiovascular reflexes

doi: 10.1101/627521

Figure Lengend Snippet: A . Representative images of DRG sections immunostained for GFRα1 and P2X5. Yellow arrows mark GFRα1 negative cells and white arrows mark positive cells. B-D . After I/R there was a significant increase in the total number of cells immunopositive for both GFRα1 and P2X5 (B) as well as total cells positive for either GFRα1 (C) or P2X5 (D) . The increase in immunopositive cells was prevented in Penα1+I/R. (n=3 per group, *p<0.05 vs Naïve and Penα1+I/R, **p<0.01 vs Naïve and Penα1+I/R; 1-way ANOVA with HSD post hoc test). E. Representative functionally identified muscle afferent obtained during ex vivo recording intracellularly filled with neurobiotin (green) was found to be immunopositive for GFRα1 (red) and P2X5 (blue) and was responsive to both “low” and “high” metabolite mixtures.

Article Snippet: Embedded DRGs were stored at −80°C, sections (15 µm) were cut on a cryostat and mounted on slides and processed for GDNF receptor GFRα1 (goat anti-GFRα1, 1:100; cat no. AF-560, R&D Systems, Minneapolis, MN, USA) and P2X5 (rabbit anti-P2X5, 1:100; cat no. APR005 Alomone Labs, Jerusalem, Israel).

Techniques: Ex Vivo

Journal: bioRxiv

Article Title: A dual role for peripheral GDNF signaling in nociception and cardiovascular reflexes

doi: 10.1101/627521

Figure Lengend Snippet: After I/R, injured muscles release a variety of metabolites that include ATP, lactate and protons that stimulate group III and IV muscle afferents. Simultaneously, GDNF is being release in the injured tissues and this induces a signaling cascade in primary sensory neurons through its receptor GFRα1 that involves increased function of CREB/ CREB binding protein (CBP), which in turn modulates the observed overexpression of ATP sensitive channel P2X5 and to a lesser degree, ASIC3. This molecular change at the DRG level in turn modulates the appearance of pain-related behaviors such as paw guarding, mechanical hypersensitivity and muscle weakness. In addition, this pathway also affects the exercise pressor reflex after injury. Targeting the overexpression of P2X5 or GFRα1 in sensory neurons or the increased GDNF levels within the muscle appears to inhibit many of these ischemic myalgia-like phenomena.

Article Snippet: Embedded DRGs were stored at −80°C, sections (15 µm) were cut on a cryostat and mounted on slides and processed for GDNF receptor GFRα1 (goat anti-GFRα1, 1:100; cat no. AF-560, R&D Systems, Minneapolis, MN, USA) and P2X5 (rabbit anti-P2X5, 1:100; cat no. APR005 Alomone Labs, Jerusalem, Israel).

Techniques: Muscles, Binding Assay, Over Expression

Journal: The Journal of Pathology

Article Title: Spatial transcriptomics exploration of the primary neuroblastoma microenvironment in archived FFPE samples unveils novel paracrine interactions

doi: 10.1002/path.6457

Figure Lengend Snippet: Identification of a spatially defined NRTN – GRFA2/RET interaction and experimental validation in NB cells. (A) Chord plot showing the number of predicted outgoing signaling interactions from the AC‐like cluster. Number of interactions with NE1 and NE2‐CA and examples of common interactions given. See supplementary material, Table for detailed results. (B) Spatial gene expression plots showing NRTN , GFRA2 , and RET expression in sections, as indicated. (C) Bar plot indicating percentages of NRTN expressing primary NB tumors with high (>90th percentile), intermediate, and low (<10th percentile) AC‐like signatures, as indicated. p values calculated using chi‐square test. (D) Kaplan–Meier survival plots comparing overall survival between NB patients with high (>90th percentile), intermediate (10th–90th percentile), and low (<10th percentile) gene expression, as indicated. p values calculated using the log rank test. (E) Western blot showing expression of GFRA2 in four different NB cell lines, as indicated. Normalized densitometry values indicated below each blot. (F, G) Time‐dependent effect of NRTN (100 ng/ml; n = 3) and/or selpercatinib (500 n m ; n = 3) application on (F) relative wound density (RWD; measured using a scratch/wound migration assay), and (G) cell growth (measured on Incucyte S3 system) on different NB cell lines, as indicated. p values for indicated timepoints calculated using an unpaired, two‐sided Student's t ‐test.

Article Snippet: Samples were subjected to western blotting analyses using the following antibodies: PITPNM3 (1:1,000) (Thermo Fisher Scientific, Cat# PA5‐21903, RRID:AB_11154802 ), GFRA2 (1:1,000) (Proteintech, Cat# 21973‐1‐AP, RRID:AB_11124728 ), and beta‐actin (1:10,000) (Cell Signaling Technology, Danvers, MA, USA; Cat# 4970, RRID:AB_2223172 ).

Techniques: Biomarker Discovery, Gene Expression, Expressing, Western Blot, Migration

Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

Article Title: Ape1/Ref-1 Stimulates GDNF/GFR?1-mediated Downstream Signaling and Neuroblastoma Proliferation

doi: 10.4196/kjpp.2009.13.5.349

Figure Lengend Snippet: GFRα1 colocalizes with a lipid raft marker in Ape1/Ref-1-expressing Neuro2a cells. (A) Neuro2a, SN4741, and SKNSH cells were transfected with empty vector (vector) or Ape1/Ref-1 expression vector (Ape1) then harvested 48 h later. Total cell extracts were prepared for immunoblotting as indicated. (B) Neuro2a cells were transfected with empty or Ape1/Ref-1 expression vector and then harvested at the indicated times after transfection. Total cell extracts were prepared for immunoblotting as indicated. (C) Neuro2a/Ape1 and vector/Neuro2a cells were immunostained using polyclonal anti-GFRα1 antibodies tagged with Alexa fluor. (D) Neuro2a/Ape1 cells were stained with anti-GFRα1 antibodies and cholera toxin (CTX), which specifically binds the lipid raft ganglioside GM1. The images show the colocalization of GFRα1 and CTX.

Article Snippet: The membranes were blocked for 1 h with TBS-t (10 mM Tris-HCl [pH 7.4], 150 mM NaCl, and 0.1% tween-20) containing 5% non-fat milk and then incubated at room temperature with primary antibodies against Ret (sc-167G), c-Src (sc-8056), Ape1/Ref-1 (sc-13104), and β-actin (sc-47778; Santa Cruz Biotechnology, Santa Cruz, CA, USA); p-Ret (Tyr905) (3221), p-AKT (Ser473) (9271), AKT (9272), p-PLC γ-1 (Tyr783) (2821) and PLCγ-1 (2822; Cell Signaling Technology, Danvers, MA, USA); and human GFRα-1 (AF714) and rat GFRα1 (MAB560; R&D Systems, Minneapolis, MN, USA).

Techniques: Marker, Expressing, Transfection, Plasmid Preparation, Western Blot, Staining

Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

Article Title: Ape1/Ref-1 Stimulates GDNF/GFR?1-mediated Downstream Signaling and Neuroblastoma Proliferation

doi: 10.4196/kjpp.2009.13.5.349

Figure Lengend Snippet: The effect of Ape1/Ref-1 on GDNF/GFRα1 signaling in Neuro2a cells. (A) Vector/Neuro2a (vector) and Neuro2a/Ape1 (Ape1) cells were treated with or without 30 ng/ml GDNF for 10 min. The cells were then lysed and subjected to immunoprecipitation (IP) with anti-Ret antibodies and analyzed for GFRα1, c-Src, phospho-Ret (pRet), and Ret by immunoblotting. (B) Vector/Neuro2a and Neuro2a/Ape1 cells were incubated with or without 30 ng/ml GDNF. At the indicated times, cell extracts were prepared and examined for activated phospho-Ret by immunoblotting.

Article Snippet: The membranes were blocked for 1 h with TBS-t (10 mM Tris-HCl [pH 7.4], 150 mM NaCl, and 0.1% tween-20) containing 5% non-fat milk and then incubated at room temperature with primary antibodies against Ret (sc-167G), c-Src (sc-8056), Ape1/Ref-1 (sc-13104), and β-actin (sc-47778; Santa Cruz Biotechnology, Santa Cruz, CA, USA); p-Ret (Tyr905) (3221), p-AKT (Ser473) (9271), AKT (9272), p-PLC γ-1 (Tyr783) (2821) and PLCγ-1 (2822; Cell Signaling Technology, Danvers, MA, USA); and human GFRα-1 (AF714) and rat GFRα1 (MAB560; R&D Systems, Minneapolis, MN, USA).

Techniques: Plasmid Preparation, Immunoprecipitation, Western Blot, Incubation

Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

Article Title: Ape1/Ref-1 Stimulates GDNF/GFR?1-mediated Downstream Signaling and Neuroblastoma Proliferation

doi: 10.4196/kjpp.2009.13.5.349

Figure Lengend Snippet: Ape1/Ref-1 increases Akt and PLCγ-1 phosphorylation in response to GDNF through GFRα1. (A) GDNF-induced Akt and PLCγ-1 phosphorylation in Ape1/Ref-1-expressing Neuro2a cells. Vector/Neuro2a and Neuro2a/Ape1 cells were incubated with or without 30 ng/ml GDNF for the indicated times and total cell extracts were prepared for immunoblotting as indicated. (B) The amounts of phospho-Akt and Phospho-PLCγ-1 were quantified by densitometry and corrected for the amount of Akt and PLCγ-1 in the corresponding lysate, rerspectively. Levels of phospho-Akt and phospho- PLCγ-1 are expressed relative to its level in non-NGEF-treated cells (0 min) transfected with control vector. The data shown are the means±S.D. from three separate experiments. (C) Neuro2a/Ape1 cells were transfected with control or GFRα1 siRNA. At 48 h after transfection, the cells were incubated with or without 30 ng/ml GDNF for the indicated times. Total cell lysates were prepared for immunoblotting as indicated. (D) The amounts of phospho-Akt and Phospho-PLCγ-1 were quantified by densitometry and corrected for the amount of Akt and PLCγ-1 in the corresponding lysate, respectively. Levels of phospho-Akt are expressed relative to its level in non-NGEF-treated cells (0 min) transfected with control siRNA. The data shown are the means±S.D. from three separate experiments.

Article Snippet: The membranes were blocked for 1 h with TBS-t (10 mM Tris-HCl [pH 7.4], 150 mM NaCl, and 0.1% tween-20) containing 5% non-fat milk and then incubated at room temperature with primary antibodies against Ret (sc-167G), c-Src (sc-8056), Ape1/Ref-1 (sc-13104), and β-actin (sc-47778; Santa Cruz Biotechnology, Santa Cruz, CA, USA); p-Ret (Tyr905) (3221), p-AKT (Ser473) (9271), AKT (9272), p-PLC γ-1 (Tyr783) (2821) and PLCγ-1 (2822; Cell Signaling Technology, Danvers, MA, USA); and human GFRα-1 (AF714) and rat GFRα1 (MAB560; R&D Systems, Minneapolis, MN, USA).

Techniques: Phospho-proteomics, Expressing, Plasmid Preparation, Incubation, Western Blot, Transfection, Control

Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

Article Title: Ape1/Ref-1 Stimulates GDNF/GFR?1-mediated Downstream Signaling and Neuroblastoma Proliferation

doi: 10.4196/kjpp.2009.13.5.349

Figure Lengend Snippet: Ape1/Ref-1 expression increases neuronal proliferation in response to GDNF. Vector/Neuro2a and Neuro2a/Ape1 cells were transfected with control (sicont) or GFRα1-siRNA (siGFRα1). At 24 h after transfection, the cells were incubated with or without 30 ng/ml GDNF for up to 48 h. The number of cells was then determined by counting every 24 h after GDNF treatment. Each value is the mean±S.D. from three separate experiments. **p<0.01.

Article Snippet: The membranes were blocked for 1 h with TBS-t (10 mM Tris-HCl [pH 7.4], 150 mM NaCl, and 0.1% tween-20) containing 5% non-fat milk and then incubated at room temperature with primary antibodies against Ret (sc-167G), c-Src (sc-8056), Ape1/Ref-1 (sc-13104), and β-actin (sc-47778; Santa Cruz Biotechnology, Santa Cruz, CA, USA); p-Ret (Tyr905) (3221), p-AKT (Ser473) (9271), AKT (9272), p-PLC γ-1 (Tyr783) (2821) and PLCγ-1 (2822; Cell Signaling Technology, Danvers, MA, USA); and human GFRα-1 (AF714) and rat GFRα1 (MAB560; R&D Systems, Minneapolis, MN, USA).

Techniques: Expressing, Plasmid Preparation, Transfection, Control, Incubation

Journal: Molecular Biology of the Cell

Article Title: Interplay of septin amphipathic helices in sensing membrane-curvature and filament bundling

doi: 10.1091/mbc.e20-05-0303

Figure Lengend Snippet: FIGURE 3: Genetic analyses of Shs1 and Cdc12 AH domains. (A) Viability of cdc12-6 cells expressing indicated SHS1 alleles expressed from the endogenous locus (with 3xHA tag, unless indicated with GFP tag, which lacks 3xHA) based on tetrad dissections at permissive temperature (24°C). Cells from genotypes labeled in blue appeared normal, without obvious septin defects. Genotypes in orange were sick, with partially or fully penetrant septin defects. Genotypes labeled in red were inviable. (B) DIC images of cdc12-6 shs1 mutants (see A) at 24°C. Scale bar, 5 μm. (C) Heterozygous diploids expressing the indicated Shs1 protein fused to GFP from the SHS1 locus. Scale bar, 5 μm. (D) Scatter plot quantifying cdc12-6-SpoVM septin complex adsorption onto different membrane curvatures. Black bars represent the mean. Error bars are the SD for more than 30 measured beads at each curvature across three replicates. Adsorption of cdc12-6-SpoVM complexes was significantly greater on a membrane curvature of 2 μm−1 than on curvatures of 0.67 and 0.4 μm−1; ** (p < 0.01 and p < 0.0001, respectively). ns, adsorption was not significantly different. (E) Western blot comparing expression of indicated Cdc12 chimeras fused to 3xHA epitope in heterozygous diploids.

Article Snippet: A polyclonal rabbit α-HA epitope antibody preparation was used at 1:2000 dilution (Rockland Immunochemicals).

Techniques: Expressing, Labeling, Adsorption, Membrane, Western Blot