Journal: PLoS ONE

Article Title: Modulation of Mouse Embryonic Stem Cell Proliferation and Neural Differentiation by the P2X7 Receptor

doi: 10.1371/journal.pone.0096281

Figure Lengend Snippet: P2X7 receptor and Oct-4 expression levels in undifferentiated (und) and cells induced to differentiation (days 0–8) were determined by ( A ) real-time PCR and ( B,C ) Western blotting assays as described in Materials and . For real-time PCR, quantitative analysis of the relative expression of P2X7R and Oct-4 in E14Tg2A cell line was performed using GAPDH mRNA transcription rates as endogenous control for normalization of expression levels. For Western blotting, P2X7R and Oct-4 expression levels were obtained and analyzed by densimetric analysis of protein bands and were compared to β-actin expression levels. The P2X7R was identified by two antibodies, which recognize the extracellular or C-terminus domain. Bars represent mean ± standard errors (S.E.) of three independent experiments performed in triplicate. Data were analyzed for statistical relevance with the One-Way ANOVA test followed by the Bonferroni post hoc test (*p<0,05,**p<0,01, ***p<0.001 compared to control data).

Article Snippet: The membranes were then incubated with primary antibodies for Oct-4 (polyclonal rabbit 1∶1000 Millipore), P2X7 receptor extracellular epitope (monoclonal rabbit 1∶2000 AbCam), P2X7 receptor C-terminus epitope (polyclonal rabbit 1∶1000 Alomone) and α-actin (1∶1000 Sigma-Aldrich) overnight at 4°C.

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

Journal: PLoS ONE

Article Title: Modulation of Mouse Embryonic Stem Cell Proliferation and Neural Differentiation by the P2X7 Receptor

doi: 10.1371/journal.pone.0096281

Figure Lengend Snippet: ( A ) P2X7 receptor isoform A, B, C and k expression in undifferentiated (UND) and cells following 8 days of neural differentiation (DIFF) was determined by RT-PCR. ( B ) P2X7R isoforms expression levels were obtained and analyzed by densimetric analysis of DNA bands and were compared to GAPDH expression levels. Data were analyzed for statistical relevance with the One-Way ANOVA test followed by the Bonferroni post hoc test (*p<0,05,**p<0,01, ***p<0.001 compared to control data).

Article Snippet: The membranes were then incubated with primary antibodies for Oct-4 (polyclonal rabbit 1∶1000 Millipore), P2X7 receptor extracellular epitope (monoclonal rabbit 1∶2000 AbCam), P2X7 receptor C-terminus epitope (polyclonal rabbit 1∶1000 Alomone) and α-actin (1∶1000 Sigma-Aldrich) overnight at 4°C.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Selective Interaction of Syntaxin 1A with KCNQ2: Possible Implications for Specific Modulation of Presynaptic Activity

doi: 10.1371/journal.pone.0006586

Figure Lengend Snippet: Values of kinetic parameters used in simulating the physiological effect of the conductances investigated in this paper.

Article Snippet: The primary antibodies used were KCNQ2-C terminus, KCNQ3-C terminus (Alomone Labs, Jerusalem, Israel) and monoclonal anti-HPC-1 (Sigma Israel, Rehovot, Israel). cDNAs and mRNAs of the chimeric channels with different transmembrane segments were described , .

Techniques:

Journal: PLoS ONE

Article Title: Selective Interaction of Syntaxin 1A with KCNQ2: Possible Implications for Specific Modulation of Presynaptic Activity

doi: 10.1371/journal.pone.0006586

Figure Lengend Snippet: A, Digitized Phosphorimager scan of SDS-PAGE analysis of [ 35 S] Met/Cys-labeled channels (KCNQ2; KCNQ2/3 and KCNQ3) and syntaxin 1A ( syx ) proteins coprecipitated by the corresponding antibodies from 1% Triton X-100 homogenates of whole oocytes, that were injected with the channels mRNA alone or with syntaxin 1A mRNA alone or coinjected with the channels and syntaxin 1A mRNAs (as indicated above the lanes). The protein samples were analyzed on an 8% gel. Arrows indicate the relevant proteins. B, KCNQ3 and KCNQ1 channels do not interact with syntaxin 1A as strongly as KCNQ2 in oocytes. Left panel : The channels and syntaxin proteins coprecipitated by the corresponding antibodies (as indicated below the lanes). Right panel : Reciprocal coimmunoprecipitation in oocytes from the same experiment, carried out using a monoclonal syntaxin 1A antibody (IP syx). C, Interaction of KCNQ2, KCNQ2\3, KCNQ3 and KCNQ1 with syntaxin 1A. Bars depict ratios (quantified by ImageQuant) of syntaxin to the channels, precipitated by the corresponding channel antibodies. Numbers in parentheses refer to number of oocyte batches. *p<0.05.

Article Snippet: The primary antibodies used were KCNQ2-C terminus, KCNQ3-C terminus (Alomone Labs, Jerusalem, Israel) and monoclonal anti-HPC-1 (Sigma Israel, Rehovot, Israel). cDNAs and mRNAs of the chimeric channels with different transmembrane segments were described , .

Techniques: SDS Page, Labeling, Injection

Journal: PLoS ONE

Article Title: Selective Interaction of Syntaxin 1A with KCNQ2: Possible Implications for Specific Modulation of Presynaptic Activity

doi: 10.1371/journal.pone.0006586

Figure Lengend Snippet: A, Syntaxin 1A (syx) reduces current amplitudes of KCNQ2 (Aa) and KCNQ2\3 (Ba) channels, expressed in Xenopus oocytes, but not those of KCNQ3* (Ca). Representative current traces from single oocytes of the same batch injected with the channels mRNA alone or with syntaxin 1A mRNA (0.75 ng/oocyte; +syx). Inset: the voltage protocol used to elicit currents. B, Syntaxin 1A reduces the maximal conductances of KCNQ2 (Ab), and KCNQ2\3 (Bb), but not of KCNQ3* (Cb). Conductance-Voltage (G–V) relationships for the channels in the presence and absence of syntaxin 1A, normalized to the maximal conductance in the absence of syntaxin 1A or each normalized to itself (inset). G values were obtained from peak currents, assuming a reversal potential of −98 mV for K+ ions. Two component Boltzmann equation G/G max = 1/(1+exp(−(V 1/2 −V)/a), ,was fitted to the data. C, Syntaxin 1A slows down only the rate of activation of KCNQ2 (Ac) but not of KCNQ2\3 (Bc) or KCNQ3* (Cc). Inset: overlay of representative traces elicited at +5 mV showing the activation of KCNQ2 currents in the presence and absence of syntaxin 1A. The rising phase of the currents elicited at all denoted potentials was fitted by a bi-exponential function, deriving fast and slow time constants (τ fast and τ slow). Data in B and C were averaged from three oocyte batches with at least 5 oocytes per batch. *p<0.05, **p<0.01.

Article Snippet: The primary antibodies used were KCNQ2-C terminus, KCNQ3-C terminus (Alomone Labs, Jerusalem, Israel) and monoclonal anti-HPC-1 (Sigma Israel, Rehovot, Israel). cDNAs and mRNAs of the chimeric channels with different transmembrane segments were described , .

Techniques: Injection, Activation Assay

Journal: PLoS ONE

Article Title: Selective Interaction of Syntaxin 1A with KCNQ2: Possible Implications for Specific Modulation of Presynaptic Activity

doi: 10.1371/journal.pone.0006586

Figure Lengend Snippet: A, The amount of KCNQ2 ( Q2 ) channel in PM is not affected by coexpression of syntaxin ( syx ). Data were obtained by measurements of confocal images in whole oocytes expressing KCNQ2 channel with external HA tag (obtained with an anti-HA antibody) or YFP tag, as indicated, alone or together with syntaxin. B, Summary of KCNQ2 PM expression and comparison of the two imaging methods. Gray bars show PM amount of KCNQ2-HA/YFP channels expressed alone. Black bars show the amount of KCNQ2-HA/YFP coexpressed with syntaxin. In both methods the PM expression level in the presence of syntaxin was normalized to the control group of oocytes expressing the channel alone. Numbers above lanes indicate the numbers of oocytes. C, The effect of coexpression of syntaxin on currents (I), corrected to the corresponding PM expression of KCNQ2-HA or KCNQ2-YFP, was measured from the same oocytes as in A. Currents were evoked by a voltage step from a holding potential of −95 mV to +5 mV and normalized to the control group of oocytes expressing the channel alone. Numbers above lanes indicate the numbers of oocytes. *p<0.05, **p<0.01. D, Syntaxin ( syx ) affects neither total protein expression nor PM content of KCNQ2. Digitized Phosphorimager scan of SDS-PAGE analysis of [ 35 S] Met/Cys-labeled KCNQ2 and syntaxin proteins, immunopurified from 110 plasma membranes (right panel; PM ) or 10 internal fractions (left panel; I ) of oocytes precipitated by KCNQ2 antibody. KCNQ2 was expressed alone ( KCNQ2 ) or together with syntaxin ( +syx ) and protein samples were analyzed on an 8% gel. E, Histogram showing normalized amount of KCNQ2 ( Q2 ; quantifies by ImageQuant) expressed with or without syntaxin, precipitated with KCNQ2 antibody from internal fractions ( I ) of oocytes. F, Histogram showing ratios (quantified by ImageQuant) of KCNQ2 amounts in plasma membranes versus internal fractions in oocytes expressing KCNQ2 alone or together with syntaxin, in the same experiments. Numbers above lanes indicate the numbers of experiments.

Article Snippet: The primary antibodies used were KCNQ2-C terminus, KCNQ3-C terminus (Alomone Labs, Jerusalem, Israel) and monoclonal anti-HPC-1 (Sigma Israel, Rehovot, Israel). cDNAs and mRNAs of the chimeric channels with different transmembrane segments were described , .

Techniques: Expressing, Imaging, SDS Page, Labeling

Journal: PLoS ONE

Article Title: Selective Interaction of Syntaxin 1A with KCNQ2: Possible Implications for Specific Modulation of Presynaptic Activity

doi: 10.1371/journal.pone.0006586

Figure Lengend Snippet: A, Schematic representation of the C-terminus of KCNQ2, in which the syntaxin 1A ( syx ) binding domain is indicated. B, Top : Interaction of syntaxin 1A with GST fusion proteins corresponding to different parts of the C-termini of KCNQ2 and KCNQ3. In vitro synthesized 35 S labeled syntaxin 1A was reacted with the indicated GST fusion proteins. Bottom : Coomassie blue (CB) staining of the protein gel. Numbers denote molecular weight markers. The bar diagram shows the normalized syntaxin 1A binding values. The intensity of the immunoreactive band of syntaxin 1A ( syx ) in different groups was normalized to the corresponding intensity of the coomassie blue ( CB ) staining of the peptide. The data were averaged from several independent experiments and includes the binding of syntaxin 1A to helices A, A+B and A+B+C. C, Top : Stochiometry of the binding of syntaxin 1A to helix A of KCNQ2, derived from binding curves that show saturation. Recombinant hexahistidine-tagged (His 6 ) cytoplasmic part of syntaxin 1A at the indicated concentrations was bound to immobilized GST-helix A (150 pmol) in a 1 ml reaction volume. Bound syntaxin 1A was determined by SDS-PAGE and immunoblotting with syntaxin 1A antibody (inset). ECL signal intensities were quantified with TINA software and converted to picomoles by the use of standard curves for the corresponding proteins. Bottom : Calibration gel which demonstrates the amount of syntaxin 1A coprecipitated in the experiment. Unbound recombinant hexahistidine-tagged (His 6 ) cytoplasmic part of syntaxin 1A at the indicated concentrations was loaded on an 8% gel and immunoblotted with syntaxin 1A antibody. D, helix A (aa 339–360) is required for syntaxin 1A's binding to KCNQ2. Oocytes were injected with syntaxin 1A mRNA alone or co-injected with syntaxin 1A and KCNQ2/Δ339–360 deletion mutant/Δ372–493 deletion mutant. The binding assay was performed as described.

Article Snippet: The primary antibodies used were KCNQ2-C terminus, KCNQ3-C terminus (Alomone Labs, Jerusalem, Israel) and monoclonal anti-HPC-1 (Sigma Israel, Rehovot, Israel). cDNAs and mRNAs of the chimeric channels with different transmembrane segments were described , .

Techniques: Binding Assay, In Vitro, Synthesized, Labeling, Staining, Molecular Weight, Derivative Assay, Recombinant, SDS Page, Western Blot, Software, Injection, Mutagenesis

Journal: PLoS ONE

Article Title: Selective Interaction of Syntaxin 1A with KCNQ2: Possible Implications for Specific Modulation of Presynaptic Activity

doi: 10.1371/journal.pone.0006586

Figure Lengend Snippet: A, Immunocytochemistry experiments show colocalization (overlay, yellow) of KCNQ2 (red) and syntaxin 1A ( syx ; green) in rat hippocampal neurons. High colocalization areas of KCNQ2 and syntaxin 1A are indicated by arrows. B, Colocalization of KCNQ2, syntaxin 1A and VAMP-2 in rat hippocampal neurons as detected by triple immunocytochemistry and illustrated by the merge images. KCNQ2 (red), syntaxin 1A (green) and VAMP-2 (blue) are indicated in the top images from left to right. The bottom images from left to right show the colocalization of KCNQ2 and VAMP-2 (merge, pink), syntaxin 1A and VAMP-2 (merge, light blue) and KCNQ2 and syntaxin 1A (merge, yellow). A varicosity colocalized with VAMP-2, syntaxin 1A and KCNQ2 is indicated by arrow. C, The same image as in B showing all three markers; KCNQ2 (red), syntaxin 1A (green) and VAMP-2 (blue). A linescan was placed through the varicosity indicated by arrow in B. The varicosity was shown to colocalize all three signals and thus, is indeed a synaptic one.

Article Snippet: The primary antibodies used were KCNQ2-C terminus, KCNQ3-C terminus (Alomone Labs, Jerusalem, Israel) and monoclonal anti-HPC-1 (Sigma Israel, Rehovot, Israel). cDNAs and mRNAs of the chimeric channels with different transmembrane segments were described , .

Techniques: Immunocytochemistry

Journal: PLoS ONE

Article Title: Selective Interaction of Syntaxin 1A with KCNQ2: Possible Implications for Specific Modulation of Presynaptic Activity

doi: 10.1371/journal.pone.0006586

Figure Lengend Snippet: A, Syntaxin 1A ( syx ) coprecipitates with KCNQ2 from 2% Chaps synaptosomal lysate by anti-KCNQ2 antibody. Lysates were incubated with KCNQ2 antibody in the absence or presence (+peptide) of antigen peptide-HC (heavy chain). Numbers indicate molecular weight markers. B, GST-syntaxin 1A fusion protein “pulls down” KCNQ2 from synaptosomal lysates. Syx-GST (corresponding to the cytosolic part of syntaxin 1A) or GST immobilized on GSH-agarose beads (each at 150 pmoles) were incubated with 2% CHAPS lysate (200 µg) for 12 h at 4°C. Precipitated proteins were separated by SDS-gel (8% polyacrylamide) and immunoblotted with anti-KCNQ2 antibody (upper panel). The lower panel shows a Ponceau S staining of the blot, which demonstrates the equal protein loading of syntaxin 1A-GST and GST proteins. C, The binding of syntaxin 1A to helices A+B+C of KCNQ2 is stronger than its binding to the same helices in KCNQ3. Syntaxin 1A coprecipitated with KCNQ2 and KCNQ3 from 2% Chaps synaptosomal lysate by anti-KCNQ2/KCNQ3 antibody. Precipitated proteins were separated by SDS-gel (8% polyacrylamide) and immunoblotted with anti-syntaxin 1A antibody.

Article Snippet: The primary antibodies used were KCNQ2-C terminus, KCNQ3-C terminus (Alomone Labs, Jerusalem, Israel) and monoclonal anti-HPC-1 (Sigma Israel, Rehovot, Israel). cDNAs and mRNAs of the chimeric channels with different transmembrane segments were described , .

Techniques: Incubation, Molecular Weight, SDS-Gel, Staining, Binding Assay

Journal: PLoS ONE

Article Title: Selective Interaction of Syntaxin 1A with KCNQ2: Possible Implications for Specific Modulation of Presynaptic Activity

doi: 10.1371/journal.pone.0006586

Figure Lengend Snippet: A, A regular firing train of action potentials generated by a 2 nA current injection into a spherical neuron containing the Hodgkin-Huxley model (current step is shown below panel c and is similar for A, B, and C). B, Similar simulation to a containing, in addition to the Hodgkin-Huxley model, also a model of the KCNQ2 as described in the Materials and Methods and at a density of 5 pS/µm 2 . The scale bar in B applies also to A, C, E, F, and G. C, Similar simulation to a containing, in addition to the Hodgkin-Huxley model, also a model of the KCNQ2+syntaxin 1A ( syx ) as described in the Materials and Methods and at a density of 2.5 pS/µm 2 . D, Simulation of the effect of KCNQ2 on the action potential ADP in a single compartment model containing the Hodgkin-Huxley model and a T-type voltage-gated Ca 2+ conductance (smooth line). Adding 5 pS/µm 2 KCNQ2 to the ADP model reduced the amplitude of the ADP (dotted line). Simulating the effect of the interaction of syntaxin 1A with KCNQ2 by using the relevant time constants from and halving the maximal conductance generated a larger ADP (dashed lines). The scale bar in D applies also to H. E, Same as in B only with KCNQ2/3. F, Same as is c only with KCNQ2/3.

Article Snippet: The primary antibodies used were KCNQ2-C terminus, KCNQ3-C terminus (Alomone Labs, Jerusalem, Israel) and monoclonal anti-HPC-1 (Sigma Israel, Rehovot, Israel). cDNAs and mRNAs of the chimeric channels with different transmembrane segments were described , .

Techniques: Generated, Injection

Journal: PLoS ONE

Article Title: Selective Interaction of Syntaxin 1A with KCNQ2: Possible Implications for Specific Modulation of Presynaptic Activity

doi: 10.1371/journal.pone.0006586

Figure Lengend Snippet: A, Digitized Phosphorimager scan of SDS-PAGE analysis of [ 35 S] Met/Cys-labeled channels (KCNQ2; KCNQ2/3 and KCNQ3) and syntaxin 1A ( syx ) proteins coprecipitated by the corresponding antibodies from 1% Triton X-100 homogenates of whole oocytes, that were injected with the channels mRNA alone or with syntaxin 1A mRNA alone or coinjected with the channels and syntaxin 1A mRNAs (as indicated above the lanes). The protein samples were analyzed on an 8% gel. Arrows indicate the relevant proteins. B, KCNQ3 and KCNQ1 channels do not interact with syntaxin 1A as strongly as KCNQ2 in oocytes. Left panel : The channels and syntaxin proteins coprecipitated by the corresponding antibodies (as indicated below the lanes). Right panel : Reciprocal coimmunoprecipitation in oocytes from the same experiment, carried out using a monoclonal syntaxin 1A antibody (IP syx). C, Interaction of KCNQ2, KCNQ2\3, KCNQ3 and KCNQ1 with syntaxin 1A. Bars depict ratios (quantified by ImageQuant) of syntaxin to the channels, precipitated by the corresponding channel antibodies. Numbers in parentheses refer to number of oocyte batches. *p<0.05.

Article Snippet: The primary antibodies used were KCNQ2-C terminus, KCNQ3-C terminus (Alomone Labs, Jerusalem, Israel) and monoclonal anti-HPC-1 (Sigma Israel, Rehovot, Israel). cDNAs and mRNAs of the chimeric channels with different transmembrane segments were described , .

Techniques: SDS Page, Labeling, Injection

Journal: PLoS ONE

Article Title: Selective Interaction of Syntaxin 1A with KCNQ2: Possible Implications for Specific Modulation of Presynaptic Activity

doi: 10.1371/journal.pone.0006586

Figure Lengend Snippet: A, Syntaxin 1A (syx) reduces current amplitudes of KCNQ2 (Aa) and KCNQ2\3 (Ba) channels, expressed in Xenopus oocytes, but not those of KCNQ3* (Ca). Representative current traces from single oocytes of the same batch injected with the channels mRNA alone or with syntaxin 1A mRNA (0.75 ng/oocyte; +syx). Inset: the voltage protocol used to elicit currents. B, Syntaxin 1A reduces the maximal conductances of KCNQ2 (Ab), and KCNQ2\3 (Bb), but not of KCNQ3* (Cb). Conductance-Voltage (G–V) relationships for the channels in the presence and absence of syntaxin 1A, normalized to the maximal conductance in the absence of syntaxin 1A or each normalized to itself (inset). G values were obtained from peak currents, assuming a reversal potential of −98 mV for K+ ions. Two component Boltzmann equation G/G max = 1/(1+exp(−(V 1/2 −V)/a), ,was fitted to the data. C, Syntaxin 1A slows down only the rate of activation of KCNQ2 (Ac) but not of KCNQ2\3 (Bc) or KCNQ3* (Cc). Inset: overlay of representative traces elicited at +5 mV showing the activation of KCNQ2 currents in the presence and absence of syntaxin 1A. The rising phase of the currents elicited at all denoted potentials was fitted by a bi-exponential function, deriving fast and slow time constants (τ fast and τ slow). Data in B and C were averaged from three oocyte batches with at least 5 oocytes per batch. *p<0.05, **p<0.01.

Article Snippet: The primary antibodies used were KCNQ2-C terminus, KCNQ3-C terminus (Alomone Labs, Jerusalem, Israel) and monoclonal anti-HPC-1 (Sigma Israel, Rehovot, Israel). cDNAs and mRNAs of the chimeric channels with different transmembrane segments were described , .

Techniques: Injection, Activation Assay

Journal: PLoS ONE

Article Title: Selective Interaction of Syntaxin 1A with KCNQ2: Possible Implications for Specific Modulation of Presynaptic Activity

doi: 10.1371/journal.pone.0006586

Figure Lengend Snippet: A, Schematic representation of the C-terminus of KCNQ2, in which the syntaxin 1A ( syx ) binding domain is indicated. B, Top : Interaction of syntaxin 1A with GST fusion proteins corresponding to different parts of the C-termini of KCNQ2 and KCNQ3. In vitro synthesized 35 S labeled syntaxin 1A was reacted with the indicated GST fusion proteins. Bottom : Coomassie blue (CB) staining of the protein gel. Numbers denote molecular weight markers. The bar diagram shows the normalized syntaxin 1A binding values. The intensity of the immunoreactive band of syntaxin 1A ( syx ) in different groups was normalized to the corresponding intensity of the coomassie blue ( CB ) staining of the peptide. The data were averaged from several independent experiments and includes the binding of syntaxin 1A to helices A, A+B and A+B+C. C, Top : Stochiometry of the binding of syntaxin 1A to helix A of KCNQ2, derived from binding curves that show saturation. Recombinant hexahistidine-tagged (His 6 ) cytoplasmic part of syntaxin 1A at the indicated concentrations was bound to immobilized GST-helix A (150 pmol) in a 1 ml reaction volume. Bound syntaxin 1A was determined by SDS-PAGE and immunoblotting with syntaxin 1A antibody (inset). ECL signal intensities were quantified with TINA software and converted to picomoles by the use of standard curves for the corresponding proteins. Bottom : Calibration gel which demonstrates the amount of syntaxin 1A coprecipitated in the experiment. Unbound recombinant hexahistidine-tagged (His 6 ) cytoplasmic part of syntaxin 1A at the indicated concentrations was loaded on an 8% gel and immunoblotted with syntaxin 1A antibody. D, helix A (aa 339–360) is required for syntaxin 1A's binding to KCNQ2. Oocytes were injected with syntaxin 1A mRNA alone or co-injected with syntaxin 1A and KCNQ2/Δ339–360 deletion mutant/Δ372–493 deletion mutant. The binding assay was performed as described.

Article Snippet: The primary antibodies used were KCNQ2-C terminus, KCNQ3-C terminus (Alomone Labs, Jerusalem, Israel) and monoclonal anti-HPC-1 (Sigma Israel, Rehovot, Israel). cDNAs and mRNAs of the chimeric channels with different transmembrane segments were described , .

Techniques: Binding Assay, In Vitro, Synthesized, Labeling, Staining, Molecular Weight, Derivative Assay, Recombinant, SDS Page, Western Blot, Software, Injection, Mutagenesis

Journal: PLoS ONE

Article Title: Selective Interaction of Syntaxin 1A with KCNQ2: Possible Implications for Specific Modulation of Presynaptic Activity

doi: 10.1371/journal.pone.0006586

Figure Lengend Snippet: A, Syntaxin 1A ( syx ) coprecipitates with KCNQ2 from 2% Chaps synaptosomal lysate by anti-KCNQ2 antibody. Lysates were incubated with KCNQ2 antibody in the absence or presence (+peptide) of antigen peptide-HC (heavy chain). Numbers indicate molecular weight markers. B, GST-syntaxin 1A fusion protein “pulls down” KCNQ2 from synaptosomal lysates. Syx-GST (corresponding to the cytosolic part of syntaxin 1A) or GST immobilized on GSH-agarose beads (each at 150 pmoles) were incubated with 2% CHAPS lysate (200 µg) for 12 h at 4°C. Precipitated proteins were separated by SDS-gel (8% polyacrylamide) and immunoblotted with anti-KCNQ2 antibody (upper panel). The lower panel shows a Ponceau S staining of the blot, which demonstrates the equal protein loading of syntaxin 1A-GST and GST proteins. C, The binding of syntaxin 1A to helices A+B+C of KCNQ2 is stronger than its binding to the same helices in KCNQ3. Syntaxin 1A coprecipitated with KCNQ2 and KCNQ3 from 2% Chaps synaptosomal lysate by anti-KCNQ2/KCNQ3 antibody. Precipitated proteins were separated by SDS-gel (8% polyacrylamide) and immunoblotted with anti-syntaxin 1A antibody.

Article Snippet: The primary antibodies used were KCNQ2-C terminus, KCNQ3-C terminus (Alomone Labs, Jerusalem, Israel) and monoclonal anti-HPC-1 (Sigma Israel, Rehovot, Israel). cDNAs and mRNAs of the chimeric channels with different transmembrane segments were described , .

Techniques: Incubation, Molecular Weight, SDS-Gel, Staining, Binding Assay

Journal: PLoS ONE

Article Title: Rod and Cone Pathway Signalling Is Altered in the P2X7 Receptor Knock Out Mouse

doi: 10.1371/journal.pone.0029990

Figure Lengend Snippet: Oligonucleotide Primer List.

Article Snippet: Rabbit anti-P2X7-receptor (C-terminus) , 1∶1000 , Alomone Labs.; Cat. No# APR 004 & Sigma; Cat No# P8232.

Techniques:

Journal: PLoS ONE

Article Title: Rod and Cone Pathway Signalling Is Altered in the P2X7 Receptor Knock Out Mouse

doi: 10.1371/journal.pone.0029990

Figure Lengend Snippet: Antibody List.

Article Snippet: Rabbit anti-P2X7-receptor (C-terminus) , 1∶1000 , Alomone Labs.; Cat. No# APR 004 & Sigma; Cat No# P8232.

Techniques: Binding Assay, Transduction

Journal: PLoS ONE

Article Title: Rod and Cone Pathway Signalling Is Altered in the P2X7 Receptor Knock Out Mouse

doi: 10.1371/journal.pone.0029990

Figure Lengend Snippet: To assess whether P2X7-R splice variants other than the full length, variant 1 are expressed in the WT and P2X7-KO mouse retina and also whether read through of variant 1 C-terminal mRNA occurs in the P2X7-KO mouse retina, PCR for (A) the central region of the P2X7-R which is common to variants 1, 2 and 3; (B) the C-terminus from within the start of the disruption in the P2X7-KO to the end of the mRNA (variant 1 only, primer pair A); and (C) the C-terminus from after the insertion deletion in the P2X7-KO mouse to the end of the receptor (variant 1 only, primer pair B) was completed. P2X7-R variant 1 C-terminal mRNA was absent (B–C) in the P2X7-KO mouse but N-terminal mRNA variants (likely variant 2 or 3) were still expressed (A). MW, molecular weight marker and the bright bar is 300 bp. (D–E) Protein analysis of P2X7-R splice variant expression was completed using Western blots of mouse retina (D) and cortex (E). An N-terminal specific P2X7-R antibody detected the protein transcript of variant 1 mRNA in WT samples 1 and 2 but not P2X7-KO samples 3 and 4 (68.41 kDa) of both retina and cortex. Protein for variant 2/3 (50.69 kDa/49.38 kDa) and 4 (17.2 kDa) was expressed in both WT and P2X7-KO retina and cortex. A monoclonal antibody against GAPDH was applied to the same blots and indicates that similar amounts of protein were loaded for WT and P2X7-KO samples.

Article Snippet: Rabbit anti-P2X7-receptor (C-terminus) , 1∶1000 , Alomone Labs.; Cat. No# APR 004 & Sigma; Cat No# P8232.

Techniques: Variant Assay, Molecular Weight, Marker, Expressing, Western Blot

Journal: PLoS ONE

Article Title: Rod and Cone Pathway Signalling Is Altered in the P2X7 Receptor Knock Out Mouse

doi: 10.1371/journal.pone.0029990

Figure Lengend Snippet: Mice were dark adapted overnight and mixed (rod and cone) ERG responses were recorded in response to full field flashes from intensity −4.2 to 2.1 log cd.s/m 2 . (A–B) Representative mixed ERG waveforms for (A) WT and (B) P2X7-KO mice are presented. (C) There was no change in the amplitude of the rod photoreceptor derived component, a-wave, as a function of intensity. (D) The amplitude of the post-photoreceptor response, the b-wave was larger in P2X7-KO mice than in WT mice across almost all intensities tested. * indicates p<0.05, a significant difference between WT and P2X7-KO.

Article Snippet: Rabbit anti-P2X7-receptor (C-terminus) , 1∶1000 , Alomone Labs.; Cat. No# APR 004 & Sigma; Cat No# P8232.

Techniques: Derivative Assay

Journal: PLoS ONE

Article Title: Rod and Cone Pathway Signalling Is Altered in the P2X7 Receptor Knock Out Mouse

doi: 10.1371/journal.pone.0029990

Figure Lengend Snippet: (A) Representative waveforms of the rod ERG recorded from WT (black) and P2X7-KO mice (grey) in response to a 2.1 log cd.s/m 2 intensity flash. The photoreceptor derived, rod PIII (modelled a-wave) was not altered in either (B) amplitude or (C) sensitivity in the P2X7-KO mouse when compared with the WT response. The post-photoreceptor, rod PII (modelled b-wave) was found to be (D) larger in amplitude and (E) faster to reach a maximum response. (F) The amplitude of the third of the inner retinal derived oscillatory potentials was significantly enhanced in the P2X7-KO mouse. * indicates p<0.05, a significant difference between WT and P2X7-KO.

Article Snippet: Rabbit anti-P2X7-receptor (C-terminus) , 1∶1000 , Alomone Labs.; Cat. No# APR 004 & Sigma; Cat No# P8232.

Techniques: Derivative Assay

Journal: PLoS ONE

Article Title: Rod and Cone Pathway Signalling Is Altered in the P2X7 Receptor Knock Out Mouse

doi: 10.1371/journal.pone.0029990

Figure Lengend Snippet: (A) Representative waveforms of the cone ERG recorded from WT (black) and P2X7-KO mice (grey) in response to a 2.1 log cd.s/m 2 intensity flash. The post-photoreceptor, cone PII (modelled b-wave) was found to be larger in (B) amplitude but not faster to reach a maximum response (C) in the P2X7-KO mouse when compared with the WT response. (D) The photopic visual acuity, assessed using the optokinetic reflex to determine the spatial frequency threshold, was higher in the P2X7-KO mouse than in the WT. * indicates p<0.05, a significant difference between WT and P2X7-KO.

Article Snippet: Rabbit anti-P2X7-receptor (C-terminus) , 1∶1000 , Alomone Labs.; Cat. No# APR 004 & Sigma; Cat No# P8232.

Techniques:

Journal: PLoS ONE

Article Title: Rod and Cone Pathway Signalling Is Altered in the P2X7 Receptor Knock Out Mouse

doi: 10.1371/journal.pone.0029990

Figure Lengend Snippet: Transverse sections of paraffin embedded retina from (A) WT and (B) P2X7-KO mice were stained with hematoxylin and eosin for comparison. The gross retinal morphology was similar in both mice. Scale bar, 20 microns.

Article Snippet: Rabbit anti-P2X7-receptor (C-terminus) , 1∶1000 , Alomone Labs.; Cat. No# APR 004 & Sigma; Cat No# P8232.

Techniques: Staining

Journal: PLoS ONE

Article Title: Rod and Cone Pathway Signalling Is Altered in the P2X7 Receptor Knock Out Mouse

doi: 10.1371/journal.pone.0029990

Figure Lengend Snippet: (A) Immunohistochemistry on a transverse retinal section from a WT mouse showing P2X7-R immunoreactivity in the outer plexiform layer (OPL), inner nuclear layer (INL), and ganglion cell layer (GCL). (C) The retina does not label with the P2X7-R antibody following incubation with P2X7-R specific peptide. Scale bar, 20 microns.

Article Snippet: Rabbit anti-P2X7-receptor (C-terminus) , 1∶1000 , Alomone Labs.; Cat. No# APR 004 & Sigma; Cat No# P8232.

Techniques: Immunohistochemistry, Incubation

Journal: PLoS ONE

Article Title: Rod and Cone Pathway Signalling Is Altered in the P2X7 Receptor Knock Out Mouse

doi: 10.1371/journal.pone.0029990

Figure Lengend Snippet: (A) P2X7-R IR (green) colocalises with horizontal cells labelled with Calbindin (red) and their terminals. (B) P2X7-R IR (green) colocalises with rod bipolar cells labelled with PKC (red). (C) Inset of rod bipolar cell terminals. (D) P2X7-R IR (green) puncta are discretely expressed within photoreceptor terminals labelled with PSD-95 (red). (E) Cone bipolar cells (Type 2 and 6) labelled with a ZNP-1 antibody (green) colocalise with P2X7-R IR (red) including both the cone bipolar cell dendrites and terminals (F). P2X7-R IR (green) colocalises with amacrine cells labelled with calretinin (G) and in particular GABAergic amacrine cells (H). (I) Ganglion cells labelled with an antibody against GFP in the Thy1-HYFP mouse (pseudo-coloured red) were P2X7-R IR (pseudo-coloured green). Scale bar, 10 microns.

Article Snippet: Rabbit anti-P2X7-receptor (C-terminus) , 1∶1000 , Alomone Labs.; Cat. No# APR 004 & Sigma; Cat No# P8232.

Techniques:

Journal: PLoS ONE

Article Title: Rod and Cone Pathway Signalling Is Altered in the P2X7 Receptor Knock Out Mouse

doi: 10.1371/journal.pone.0029990

Figure Lengend Snippet: (A) P2X7-R IR (red) is associated with but does not colocalise with ribbon synapses (ribeye, green) on bipolar cell terminals (VGLUT, blue). (B) Magnified inset from A, arrows indicate ribbon synapses associated with P2X7-R IR puncta. (C) P2X7-R IR (red) colocalises with conventional synapses (bassoon, green) distinct from bipolar cell terminals (VGLUT, blue). (D) Magnified inset from C, arrows indicate conventional synapses that colocalise with P2X7-R IR puncta. Co-localisation analysis indicates that P2X7-R IR puncta do not co-localise with ribbon synapses (E) but do show significant colocalisation with conventional synapses (F) in the IPL. Scale bars are 5 microns for A & C and 2 microns for B & D.

Article Snippet: Rabbit anti-P2X7-receptor (C-terminus) , 1∶1000 , Alomone Labs.; Cat. No# APR 004 & Sigma; Cat No# P8232.

Techniques:

Journal: PLoS ONE

Article Title: Rod and Cone Pathway Signalling Is Altered in the P2X7 Receptor Knock Out Mouse

doi: 10.1371/journal.pone.0029990

Figure Lengend Snippet: Ribbons are indicated by arrows and labelled amacine cells are indicated by astericks. (A), (B) & (C) P2X7-R immunoreactivity labels amacrine cells adjacent to rod bipolar cell ribbon synapses in the IPL. (D) A conventional amacrine cell synapse is labelled for P2X7 immunoreactivity in close proximity to a rod bipolar cell terminal. Scale bars are 200 nm for A & B and 500 nm for C & D.

Article Snippet: Rabbit anti-P2X7-receptor (C-terminus) , 1∶1000 , Alomone Labs.; Cat. No# APR 004 & Sigma; Cat No# P8232.

Techniques:

Journal: PLoS ONE

Article Title: Rod and Cone Pathway Signalling Is Altered in the P2X7 Receptor Knock Out Mouse

doi: 10.1371/journal.pone.0029990

Figure Lengend Snippet: (A–B) Müller cells, labelled with an antibody against glutamine synthetise (red) were found to be closely associated with P2X7-R-IR puncta in the IPL (green), but not co-localised in the IPL. (C) Astrocytes, labelled with glial fibrilliary acidic protein (GFAP; red) and P2X7-R-IR (green) were found to co-localise in retinal flat mount. (D) Microglia labelled with mouse anti-GFP (pseudo-coloured red) from CX3CR1-GFP heterozygous mice were also found to co-localise with P2X7-R-IR (pseudo-coloured green). Scale bar, 10 microns.

Article Snippet: Rabbit anti-P2X7-receptor (C-terminus) , 1∶1000 , Alomone Labs.; Cat. No# APR 004 & Sigma; Cat No# P8232.

Techniques:

Journal: PLoS ONE

Article Title: Direct Interaction of Endogenous Kv Channels with Syntaxin Enhances Exocytosis by Neuroendocrine Cells

doi: 10.1371/journal.pone.0001381

Figure Lengend Snippet: A. A schematic presentation of the Kv2.1 channel showing fragments generated as GST fusion proteins. The grey box on the left, the 6 dark boxes and the grey box on the right denote the N terminus, the 6 trans membrane and the C terminus domains, respectively. Numbers denote amino acids. B. GST fusion proteins corresponding to Kv2.1 C terminus but not N terminus pull down syntaxin from PC12 cell lysate. Proteins (each at 150 pmol) immobilized on glutathione affinity beads were incubated with 10 6 cells in K-Glu buffer with 1% Triton X-100 for 12 hr at 4°C. Precipitated proteins were separated by SDS-PAGE (12% polyacrylamide) and immunobloted with syntaxin antibody ( IB Syx ), or stained with Ponceau S (lower panel). PC12 lysates (0.5% of that processed in the pull down reaction) were loaded for reference. Right and left panels are two separate experiments. Molecular weights are marked on the right. C. GST-fused Kv2.1-C1a protein or GST alone (150 pmole each) immobilized on glutathione affinity beads were incubated with 35 S-labeled synaptotagmin ( Syt ), 35 S-labeled syntaxin ( Syx ), 35 S-labeled SNAP-25 or 35 S-labeled VAMP2 for 1 h. in 1 ml 0.1% Triton X-100. Gluthation-eluted proteins ( right panel ) and input (20 µl out of 1000 µl of reaction mixture taken before the addition of beads; left panel ) were analyzed by SDS-PAGE.

Article Snippet: The primary antibodies used were anti Kv1.1-C terminus and Kv2.1-C terminus (Alomone Labs Jerusalem, Israel), monoclonal anti HPC-1 (Sigma Israel, Rehovot), monoclonal anti SNAP-25 (BD Transduction Lab, Lexington, KY), The DNAs of Kv1.1 and Kv2.1 fragments for production of GST fusion proteins were constructed as described previously ( , .

Techniques: Generated, Incubation, SDS Page, Staining, Labeling

Journal: PLoS ONE

Article Title: Direct Interaction of Endogenous Kv Channels with Syntaxin Enhances Exocytosis by Neuroendocrine Cells

doi: 10.1371/journal.pone.0001381

Figure Lengend Snippet: (A) PC12 cells preloaded with [ 3 H]-NE and gently homogenized ( “cracked” cells), either underwent 15 min incubation in the presence of 2 mM MgATP and brain cytosol at 30°C (“priming”) or not, followed by either 15 min incubation with added 1.6 mM Ca 2+ (1 µM free Ca 2+ ; at 30° (“triggering”) or not, as indicated below the bars. Cells were pelleted and the secreted [ 3 H]-NE in the supernatant was quantified by scintillation counting and expressed as a percentage of the total [ 3 H]-NE in the cell. 10 6 cells per reaction were used. The difference between the amounts of [ 3 H]-NE secreted in the two reactions on the two bars on the right was defined and is referred to hereafter as the Ca 2+ -triggered release. (B) Cracked cells preloaded with [ 3 H]-NE underwent priming and triggering reactions in the absence or presence of 10–20 µM GST-fusion peptides corresponding to cytosolic parts of Kv2.1 and Kv1.1 (shown in ) or to GST itself (as indicated below bars). Values of Ca 2+ -triggered release measured in the presence of the peptides (see ) were normalized to the control release determined in the absence of peptides (defined as 100%). Each bar in A and B depicts the mean±s.e.m from several independent experiments (numbers of experiments are in parentheses above bars). **, p <0.001 (compared with GST). (C) Cells were stimulated to release NE in the presence of increasing concentrations of GST-fused Kv2.1-C1 protein. Each point in the curve represents mean±s.e.m values from several independent experiments (numbers of experiments in parentheses above bars). **, p<0.001 (compared with 10 nM GST-C1).

Article Snippet: The primary antibodies used were anti Kv1.1-C terminus and Kv2.1-C terminus (Alomone Labs Jerusalem, Israel), monoclonal anti HPC-1 (Sigma Israel, Rehovot), monoclonal anti SNAP-25 (BD Transduction Lab, Lexington, KY), The DNAs of Kv1.1 and Kv2.1 fragments for production of GST fusion proteins were constructed as described previously ( , .

Techniques: Incubation

Journal: PLoS ONE

Article Title: Direct Interaction of Endogenous Kv Channels with Syntaxin Enhances Exocytosis by Neuroendocrine Cells

doi: 10.1371/journal.pone.0001381

Figure Lengend Snippet: (A) PC12 cells preloaded with [ 3 H]-NE and gently homogenized ( “cracked” cells), either underwent 15 min incubation in the presence of 2 mM MgATP and brain cytosol at 30°C (“priming”) or not, followed by either 15 min incubation with added 1.6 mM Ca 2+ (1 µM free Ca 2+ ; at 30° (“triggering”) or not, as indicated below the bars. Cells were pelleted and the secreted [ 3 H]-NE in the supernatant was quantified by scintillation counting and expressed as a percentage of the total [ 3 H]-NE in the cell. 10 6 cells per reaction were used. The difference between the amounts of [ 3 H]-NE secreted in the two reactions on the two bars on the right was defined and is referred to hereafter as the Ca 2+ -triggered release. (B) Cracked cells preloaded with [ 3 H]-NE underwent priming and triggering reactions in the absence or presence of 10–20 µM GST-fusion peptides corresponding to cytosolic parts of Kv2.1 and Kv1.1 (shown in ) or to GST itself (as indicated below bars). Values of Ca 2+ -triggered release measured in the presence of the peptides (see ) were normalized to the control release determined in the absence of peptides (defined as 100%). Each bar in A and B depicts the mean±s.e.m from several independent experiments (numbers of experiments are in parentheses above bars). **, p <0.001 (compared with GST). (C) Cells were stimulated to release NE in the presence of increasing concentrations of GST-fused Kv2.1-C1 protein. Each point in the curve represents mean±s.e.m values from several independent experiments (numbers of experiments in parentheses above bars). **, p<0.001 (compared with 10 nM GST-C1).

Article Snippet: The primary antibodies used were anti Kv1.1-C terminus and Kv2.1-C terminus (Alomone Labs Jerusalem, Israel), monoclonal anti HPC-1 (Sigma Israel, Rehovot), monoclonal anti SNAP-25 (BD Transduction Lab, Lexington, KY), The DNAs of Kv1.1 and Kv2.1 fragments for production of GST fusion proteins were constructed as described previously ( , .

Techniques: Incubation