Journal: eBioMedicine

Article Title: Neural synaptic vesicle autoimmunity following aerosolized porcine neural tissue exposure: insights into autoimmune inflammatory polyradiculoneuropathy

doi: 10.1016/j.ebiom.2025.106053

Figure Lengend Snippet: Confirmation of synaptophysin- and GAP43-IgG in patients with OIPN by ELISA and CBA . a) Fifteen of 20 patients with OIPN sera tested positive for synaptophysin-IgG by ELISA compared to none of the control cohorts. b) Representative immunofluorescent images of IgG binding to GFP-tagged synaptophysin-transfected COS7 cells. Commercial synaptophysin-IgG and OIPN-IgG (magenta) co-localises (white in merge) with GFP-tagged synaptophysin protein (green). c) Twelve of 20 sera from patients with OIPN tested positive for GAP43-IgG by ELISA. d) Representative immunofluorescent images of IgG binding (magenta) to GFP-tagged GAP43 transfected COS7 cells. Colocalisation (white in merge) is observed for commercial antibody and OIPN-IgG with GFP-GAP43 protein. b and d) DNA is labelled with DAPI, blue. Healthy adult human IgG fails to bind to transfected cells. Note: No IgG bound to non-transfected cells (solitary blue nuclei). Scale bars, 20 μm. Key: OIPN, occupational inflammatory polyradiculoneuropathy; ELISA, enzyme-linked immunosorbent assay; GFP, green fluorescence protein; DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: Immulon 2HB plates were coated (10 ng/well) with PhIP-Seq-identified antigens: synaptophysin peptide 224-313 (Mayo Clinic Proteomics Core Facility) or GAP43 recombinant protein (Novus, #NBP2-53033) diluted in 0.01 M PBS, pH 7.4.

Techniques: Enzyme-linked Immunosorbent Assay, Control, Binding Assay, Transfection, Fluorescence

Journal:

Article Title: Modulation of Astrocyte P2Y 1 Receptors by the Carboxyl Terminal Domain of the Gap Junction Protein Cx43

doi: 10.1002/glia.20598

Figure Lengend Snippet: (A) Western blot showing expression of Cx43 in WT (lane 1) and in Cx43-null astrocytes transfected with full length Cx43 (lane 3), with Cx43 carboxyl terminus (lane 4) and with Cx43M257 (lane 5). Lane 2 corresponds to untransfected Cx43-null cells. Lanes 1–4 correspond to immunoblots performed with Cx43-18A antibody and lane 5 with Cx43-16A. (B) Time courses of calcein FRAP obtained from untreated (black squares) and 48 h carbenoxolone-treated (open triangles) WT astrocytes, and from untransfected (open circles) and transfected Cx43-null astrocytes with full length Cx43 (open squares) and Cx43M257 (black circles). Data were obtained from astrocytes cultured from a minimum of 3 litters of mice.

Article Snippet: The following primary antibodies were employed: polyclonal antiP2Y1 (1:200; Alomone Labs), and two different polyclonal antiCx43 antibodies (gifts from Dr. E.L. Hertzberg, Albert Einstein College of Medicine), one recognizing Cx43 at position 241–261 (16A) and the other at position 345–360 (18A).

Techniques: Western Blot, Expressing, Transfection, Cell Culture

Journal:

Article Title: Modulation of Astrocyte P2Y 1 Receptors by the Carboxyl Terminal Domain of the Gap Junction Protein Cx43

doi: 10.1002/glia.20598

Figure Lengend Snippet: (A) Dose-response curves obtained for untreated (black squares) and 48 h carbenoxolone-treated (open triangles) WT and untransfected Cx43-null spinal cord astrocytes exposed to the P2Y1R agonist 2-MeS-ATP. Note that blockade of gap junctional communication in WT astrocytes does not alter the half-maximal response (EC50 value) induced by the P2Y1R agonist. Mean values were obtained from four to seven independent experiments. (B) Dose-response curves obtained for WT (black squares), untransfected (open circles) and transfected Cx43-null astrocytes with full length Cx43 (open squares), Cx43M257 (black circles) and Cx43CT (black triangles). Note that both full length Cx43 and Cx43CT but not Cx43M257 shifted the EC50 values of 2-MeS-ATP obtained for Cx43-null astrocytes to those obtained in WT cells. Data were obtained from 5 to 8 litters.

Article Snippet: The following primary antibodies were employed: polyclonal antiP2Y1 (1:200; Alomone Labs), and two different polyclonal antiCx43 antibodies (gifts from Dr. E.L. Hertzberg, Albert Einstein College of Medicine), one recognizing Cx43 at position 241–261 (16A) and the other at position 345–360 (18A).

Techniques: Transfection

Journal:

Article Title: Modulation of Astrocyte P2Y 1 Receptors by the Carboxyl Terminal Domain of the Gap Junction Protein Cx43

doi: 10.1002/glia.20598

Figure Lengend Snippet: Bar histograms showing the mean values of P2Y1R expression levels in WT, untransfected Cx43-null (KO), and in Cx43-null transfected with Cx43 CT, Cx43 truncated at position 257 (M257) and with full length Cx43. An example of western blots for Cx43 and β-actin is shown above.

Article Snippet: The following primary antibodies were employed: polyclonal antiP2Y1 (1:200; Alomone Labs), and two different polyclonal antiCx43 antibodies (gifts from Dr. E.L. Hertzberg, Albert Einstein College of Medicine), one recognizing Cx43 at position 241–261 (16A) and the other at position 345–360 (18A).

Techniques: Expressing, Transfection, Western Blot

Journal:

Article Title: Modulation of Astrocyte P2Y 1 Receptors by the Carboxyl Terminal Domain of the Gap Junction Protein Cx43

doi: 10.1002/glia.20598

Figure Lengend Snippet: (A) Dose-response curves obtained for WT (black squares) and untransfected (open circles) and Cx43Δ260–280 transfected (black triangles) Cx43-null astrocytes exposed to 2-MeS-ATP, showing that deletion of the Cx43 SH3 domain does not rescue P2Y1 receptor function. Mean±SE values are from 100 to 200 cells obtained from three litters. (B) Dose-response curves obtained from WT (black squares), and Cx43-null astrocytes untreated (open circles) and treated (black triangles) with a membrane permeant peptide corresponding to amino acids 260–280 of Cx43CT. (C) Bar histograms of the mean±SE values of intracellular calcium mobilization induced by 100 nM 2-MeS-ATP recorded from WT, Cx43-null and Cx43-null transfected with Cx43CT and Cx43M257 in the absence and presence of 5 µM PP2. Note that only untransfected and Cx43M257 transfected Cx43-null astrocytes that were not exposed to PP2 did not respond to agonist with intracellular calcium levels similar to WT astrocytes. Mean ± SE values are from 4 litters (**P < 0.001; ANOVA followed by Newman-Keuls’ Multiple Comparison Test).

Article Snippet: The following primary antibodies were employed: polyclonal antiP2Y1 (1:200; Alomone Labs), and two different polyclonal antiCx43 antibodies (gifts from Dr. E.L. Hertzberg, Albert Einstein College of Medicine), one recognizing Cx43 at position 241–261 (16A) and the other at position 345–360 (18A).

Techniques: Transfection

Journal:

Article Title: Modulation of Astrocyte P2Y 1 Receptors by the Carboxyl Terminal Domain of the Gap Junction Protein Cx43

doi: 10.1002/glia.20598

Figure Lengend Snippet: (A) Western blot showing decreased expression levels of Cx43 following exposure of spinal cord astrocytes to IL-1β (B) Dose-response curves obtained for 2-MeS-ATP performed on Fura-2 loaded WT and Cx43 KO astrocytes treated for 24 h with IL-1β (20 ng/mL). Note that exposure to the cytokine altered the agonist EC50 values in WT astrocytes. About 180 cells from three independent experiments were used in each condition.

Article Snippet: The following primary antibodies were employed: polyclonal antiP2Y1 (1:200; Alomone Labs), and two different polyclonal antiCx43 antibodies (gifts from Dr. E.L. Hertzberg, Albert Einstein College of Medicine), one recognizing Cx43 at position 241–261 (16A) and the other at position 345–360 (18A).

Techniques: Western Blot, Expressing

Journal: Frontiers in Cellular Neuroscience

Article Title: Hyperpolarization-Activated Cyclic Nucleotide-Gated Ion (HCN) Channels Regulate PC12 Cell Differentiation Toward Sympathetic Neuron

doi: 10.3389/fncel.2019.00415

Figure Lengend Snippet: NGF-induced PC12 cell differentiation toward sympathetic neurons and expressions of GAP-43 and TH. (A) Immunofluorescent stains of GAP-43 (red, upper row) and TH (red, lower row). Nuclei were stained blue by DAPI. Note that NGF induced significant neurite outgrowth. Scale bar, 10 μm. (B,C) Real-time PCR and western blotting results of GAP-43 and TH expressions in PC12 cells treated or untreated with NGF. ∗∗∗ p < 0.001 vs. respective control. ### p < 0.001 vs. respective control; n = 3 for each value.

Article Snippet: The membranes were blocked overnight with 5% BSA and then were respectively incubated with polyclonal anti-HCN1 (APC-056), anti-HCN2 (APC-030), anti-HCN3 (APC-057), anti-HCN4 (AGP-004) at 1:1000 dilution (Alomone Labs, Jerusalem, Israel), or with anti-GAP-43 (sc-135915) or anti-TH (sc-136100) at 1:1000 dilution (Santa Cruz Biotechnology, Inc. United States) overnight at 4°C.

Techniques: Cell Differentiation, Staining, Real-time Polymerase Chain Reaction, Western Blot

Journal: Frontiers in Cellular Neuroscience

Article Title: Hyperpolarization-Activated Cyclic Nucleotide-Gated Ion (HCN) Channels Regulate PC12 Cell Differentiation Toward Sympathetic Neuron

doi: 10.3389/fncel.2019.00415

Figure Lengend Snippet: The suppressing effects of ivabradine (IVA) on the mRNA and protein expressions of HCN channel isoforms and GAP-43 and the neurite outgrowth. (A–D) Real-time PCR and western blotting results of the mRNA and protein expression levels respectively for HCN1-4 isoforms in NGF-treated PC12 cells relative to GADPH level. ∗∗ p < 0.01, ∗∗∗ p < 0.001 vs. NGF treatment alone in the mRNA level. # p < 0.05, ## p < 0.01 vs. NGF treatment alone in the protein level. (E) Representative western blotting bands ( upper ) and the mRNA and protein semiquantitative values ( lower ) of GAP-43 in NGF-treated PC12 cells. ∗∗ p < 0.01, ∗∗∗ p < 0.001 vs. NGF treatment alone in the mRNA level. # p < 0.05, ### p < 0.001 vs. NGF treatment alone in the protein level. (F) Immunofluorescent stains of GAP-43 protein (red), displaying that NGF induced, while ivabradine inhibited, the neurite outgrowth in differentiating PC12 cells. DAPI was used for cell nuclei staining (blue). Scale bar, 10 μm. (G,H) Quantitative morphological parameters of neurite outgrowth including the total neurite length and the maximal neurite length (longest process per cell). Results are calculated from three independent experiments and presented as mean ± SEM, 96 < n < 213, ∗ p < 0.05, ∗∗ p < 0.01 vs. NGF treatment alone.

Article Snippet: The membranes were blocked overnight with 5% BSA and then were respectively incubated with polyclonal anti-HCN1 (APC-056), anti-HCN2 (APC-030), anti-HCN3 (APC-057), anti-HCN4 (AGP-004) at 1:1000 dilution (Alomone Labs, Jerusalem, Israel), or with anti-GAP-43 (sc-135915) or anti-TH (sc-136100) at 1:1000 dilution (Santa Cruz Biotechnology, Inc. United States) overnight at 4°C.

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

Journal: Frontiers in Cellular Neuroscience

Article Title: Hyperpolarization-Activated Cyclic Nucleotide-Gated Ion (HCN) Channels Regulate PC12 Cell Differentiation Toward Sympathetic Neuron

doi: 10.3389/fncel.2019.00415

Figure Lengend Snippet: Knocking-down HCN2 and HCN4 expression with siRNAs inhibited GAP-43 expression and neurite outgrowth in PC12 cells. NGF-treated PC12 cells were transiently transfected with the negative control siRNA (NC-siRNA) or with the siRNA respectively for HCN1, HCN2, HCN3 or HCN4 using Lipofectamine ® RNAiMAX Reagent for 48 h before cells were harvested for detection. (A–D) The mRNA and protein expressions of HCN1-4 isoforms assayed by qPCR and western blotting in comparison to GADPH in NGF-treated PC12 cells. ∗∗ p < 0.01, ∗∗∗ p < 0.001 vs. negative control siRNA (N.C.) in the mRNA level. # p < 0.05, ## p < 0.01, ### p < 0.001 vs. N.C. in the protein level. (E) Representative western blotting results ( upper ) and the densitometric analysis of GAP-43 mRNA and protein expressions ( lower ) ( n = 3–6 independent experiments). ∗ p < 0.05, ∗∗∗ p < 0.001 vs. N.C. in the mRNA level. ### p < 0.001 vs. N.C. in the protein level. (F) Immunofluorescent stains of GAP-43 in PC12 cells transiently transfected with siRNA targeting HCN2 or HCN4. Scale bar, 10 μm. (G,H) Quantification of neurite morphological parameters (total length of neurite outgrowth and longest process per cell). Results are calculated from three independent experiments and presented as mean ± SEM, 50 < n < 84, ∗ p < 0.05, ∗∗ p < 0.01 vs. N.C.

Article Snippet: The membranes were blocked overnight with 5% BSA and then were respectively incubated with polyclonal anti-HCN1 (APC-056), anti-HCN2 (APC-030), anti-HCN3 (APC-057), anti-HCN4 (AGP-004) at 1:1000 dilution (Alomone Labs, Jerusalem, Israel), or with anti-GAP-43 (sc-135915) or anti-TH (sc-136100) at 1:1000 dilution (Santa Cruz Biotechnology, Inc. United States) overnight at 4°C.

Techniques: Expressing, Transfection, Negative Control, Western Blot

Journal: Frontiers in Cellular Neuroscience

Article Title: Hyperpolarization-Activated Cyclic Nucleotide-Gated Ion (HCN) Channels Regulate PC12 Cell Differentiation Toward Sympathetic Neuron

doi: 10.3389/fncel.2019.00415

Figure Lengend Snippet: Overexpression of HCN2 and HCN4 enhanced GAP-43 expression and neurite outgrowth in PC12 cells. Results were obtained 48 h after cell transfection with either empty plasmid pcDNA3.0/vector or pcDNA3.0/HCN2 or pcDNA3.0/HCN4. (A,B) Representative western blots ( upper ) and semiquantitative values ( lower ) of the mRNA and protein levels of HCN2 and HCN4 isoforms. ∗∗ p < 0.01, ∗∗∗ p < 0.001 vs. control (pcDNA3.0/vector) in the mRNA level. # p < 0.05 vs. control (pcDNA3.0/vector) in the protein level, n = 4−6 independent experiments. (C) qPCR and western blotting results showing the effects of HCN2 or HCN4 overexpression on the mRNA and protein expressions of GAP-43. ∗∗∗ p < 0.001 vs. empty plasmid in the mRNA level. ### p < 0.001 vs. empty plasmid in the protein level, n = 4−6 independent experiments. (D) Immunofluorescent stains of GAP-43 (red) of PC12 cells transiently transfected respectively with pcDNA3.0/vector, pcDNA3.0/HCN2 or pcDNA3.0/HCN4 plasmids. Scale bar, 10 μm. (E,F) Quantification of neurite morphological parameters including total neurite outgrowth and longest process per cell. Results are calculated from three independent experiments and presented as mean ± SEM, 50 < n < 90, ∗ p < 0.05 vs. empty vector.

Article Snippet: The membranes were blocked overnight with 5% BSA and then were respectively incubated with polyclonal anti-HCN1 (APC-056), anti-HCN2 (APC-030), anti-HCN3 (APC-057), anti-HCN4 (AGP-004) at 1:1000 dilution (Alomone Labs, Jerusalem, Israel), or with anti-GAP-43 (sc-135915) or anti-TH (sc-136100) at 1:1000 dilution (Santa Cruz Biotechnology, Inc. United States) overnight at 4°C.

Techniques: Over Expression, Expressing, Transfection, Plasmid Preparation, Western Blot