Structured Review

Ipsen Group kv7 2
NP1 interacts and colocalizes with <t>Kv7.2</t> at presynaptic terminals of excitatory synapses and axonal growth cones. A – D , Representative Western blots of immunoprecipitation eluates separated in 6% Tris-Glycine (for high molecular weight proteins) and 4–12% Bis-Tris gels (for low molecular weight proteins). TL, Total protein lysate; Syx, syntaxin. A , Both native Kv7.2 and syntaxin 1A, but not Kv7.3, coprecipitate with NP1 in total brain extracts. B , Both native Kv7.2 and NP1 coprecipitate with syntaxin in total brain extracts. C , D , Recombinant NP1 coprecipitates Kv7.2, but not syntaxin or Kv7.3, in 293T cells transfected with NP1, 5Myc-Kv7.2, 2HA-Kv7.3, and syntaxin 1A cDNAs. Kv7.2 and Kv7.3 were immunoprecipitated with antibodies against their respective Myc and HA tags. E , F , Immunofluorescence studies and confocal microscopy were performed in high-density ( E ) or low-density ( F ) isolated cortical neurons. E , Top, Confocal sections of 0.772 μm in the z -plane showing immunofluorescence of NP1, VGLUT1, Kv7.2, and negative control (omitting primary antibodies). Bottom, Colocalization (in white) of the excitatory presynaptic marker VGLUT1 (blue) with NP1 (green) and Kv7.2 (red) is shown in a single section with the corresponding orthogonal views of the stack of confocal sections. White arrows indicate sites of colocalization. F , NP1 (green) and KV7.2 (magenta) immunofluorescence and DIC images of an isolated cortical cultured neuron (1×) with its corresponding axonal growth cone highlighted in a white square box, shown in a confocal section of 0.772 μm in the z -plane at higher (5×) magnification. The negative control for primary antibodies is shown in another growth cone on the right. The image in the bottom is the merge of NP1 and Kv7.2 immunofluorescence images in the single confocal section obtained at 5× showing colocalization (white) of NP1 and Kv7.2 in the growth cone, with the corresponding orthogonal views from its respective stack of confocal sections. Images were acquired using restricted spectral emission wavelength ranges chosen to avoid crosstalk or bleed-through between the three different channels. Scale bar, 5 μm.
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Images

1) Product Images from "Neuronal Pentraxin 1 Negatively Regulates Excitatory Synapse Density and Synaptic Plasticity"

Article Title: Neuronal Pentraxin 1 Negatively Regulates Excitatory Synapse Density and Synaptic Plasticity

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.2548-14.2015

NP1 interacts and colocalizes with Kv7.2 at presynaptic terminals of excitatory synapses and axonal growth cones. A – D , Representative Western blots of immunoprecipitation eluates separated in 6% Tris-Glycine (for high molecular weight proteins) and 4–12% Bis-Tris gels (for low molecular weight proteins). TL, Total protein lysate; Syx, syntaxin. A , Both native Kv7.2 and syntaxin 1A, but not Kv7.3, coprecipitate with NP1 in total brain extracts. B , Both native Kv7.2 and NP1 coprecipitate with syntaxin in total brain extracts. C , D , Recombinant NP1 coprecipitates Kv7.2, but not syntaxin or Kv7.3, in 293T cells transfected with NP1, 5Myc-Kv7.2, 2HA-Kv7.3, and syntaxin 1A cDNAs. Kv7.2 and Kv7.3 were immunoprecipitated with antibodies against their respective Myc and HA tags. E , F , Immunofluorescence studies and confocal microscopy were performed in high-density ( E ) or low-density ( F ) isolated cortical neurons. E , Top, Confocal sections of 0.772 μm in the z -plane showing immunofluorescence of NP1, VGLUT1, Kv7.2, and negative control (omitting primary antibodies). Bottom, Colocalization (in white) of the excitatory presynaptic marker VGLUT1 (blue) with NP1 (green) and Kv7.2 (red) is shown in a single section with the corresponding orthogonal views of the stack of confocal sections. White arrows indicate sites of colocalization. F , NP1 (green) and KV7.2 (magenta) immunofluorescence and DIC images of an isolated cortical cultured neuron (1×) with its corresponding axonal growth cone highlighted in a white square box, shown in a confocal section of 0.772 μm in the z -plane at higher (5×) magnification. The negative control for primary antibodies is shown in another growth cone on the right. The image in the bottom is the merge of NP1 and Kv7.2 immunofluorescence images in the single confocal section obtained at 5× showing colocalization (white) of NP1 and Kv7.2 in the growth cone, with the corresponding orthogonal views from its respective stack of confocal sections. Images were acquired using restricted spectral emission wavelength ranges chosen to avoid crosstalk or bleed-through between the three different channels. Scale bar, 5 μm.
Figure Legend Snippet: NP1 interacts and colocalizes with Kv7.2 at presynaptic terminals of excitatory synapses and axonal growth cones. A – D , Representative Western blots of immunoprecipitation eluates separated in 6% Tris-Glycine (for high molecular weight proteins) and 4–12% Bis-Tris gels (for low molecular weight proteins). TL, Total protein lysate; Syx, syntaxin. A , Both native Kv7.2 and syntaxin 1A, but not Kv7.3, coprecipitate with NP1 in total brain extracts. B , Both native Kv7.2 and NP1 coprecipitate with syntaxin in total brain extracts. C , D , Recombinant NP1 coprecipitates Kv7.2, but not syntaxin or Kv7.3, in 293T cells transfected with NP1, 5Myc-Kv7.2, 2HA-Kv7.3, and syntaxin 1A cDNAs. Kv7.2 and Kv7.3 were immunoprecipitated with antibodies against their respective Myc and HA tags. E , F , Immunofluorescence studies and confocal microscopy were performed in high-density ( E ) or low-density ( F ) isolated cortical neurons. E , Top, Confocal sections of 0.772 μm in the z -plane showing immunofluorescence of NP1, VGLUT1, Kv7.2, and negative control (omitting primary antibodies). Bottom, Colocalization (in white) of the excitatory presynaptic marker VGLUT1 (blue) with NP1 (green) and Kv7.2 (red) is shown in a single section with the corresponding orthogonal views of the stack of confocal sections. White arrows indicate sites of colocalization. F , NP1 (green) and KV7.2 (magenta) immunofluorescence and DIC images of an isolated cortical cultured neuron (1×) with its corresponding axonal growth cone highlighted in a white square box, shown in a confocal section of 0.772 μm in the z -plane at higher (5×) magnification. The negative control for primary antibodies is shown in another growth cone on the right. The image in the bottom is the merge of NP1 and Kv7.2 immunofluorescence images in the single confocal section obtained at 5× showing colocalization (white) of NP1 and Kv7.2 in the growth cone, with the corresponding orthogonal views from its respective stack of confocal sections. Images were acquired using restricted spectral emission wavelength ranges chosen to avoid crosstalk or bleed-through between the three different channels. Scale bar, 5 μm.

Techniques Used: Western Blot, Immunoprecipitation, Molecular Weight, Recombinant, Transfection, Immunofluorescence, Confocal Microscopy, Isolation, Negative Control, Marker, Cell Culture

Knockdown of NP1 reduces Kv7.2 surface expression and M-current. A , B , The proportion of Kv7.2 at the neuronal surface was determined by biotinylation (30 min at 4°C) of all surface proteins in cultured cortical neurons. The ratio of the surface over the total amount of receptor was determined by densitometric analysis and expressed as a percentage of control values. Only samples of biotinylated proteins lacking immunoreactivity to actin, which indicates contamination with intracellular proteins, were used for analyses. A , Representative Western blots of Kv7.2, Na-KATPase, and actin from total (T) and biotinylated (B) proteins. B , Quantitative analysis of the effect of gain and loss of NP1 on the surface/total levels of the Kv7.2 subunit of the voltage-gated Kv7/M K + channels. Values are mean ± SEM of shRdm ( n = 11), shNP1 ( n = 11), and pWPI-NP1 ( n = 5), from at least three independent experiments; * p
Figure Legend Snippet: Knockdown of NP1 reduces Kv7.2 surface expression and M-current. A , B , The proportion of Kv7.2 at the neuronal surface was determined by biotinylation (30 min at 4°C) of all surface proteins in cultured cortical neurons. The ratio of the surface over the total amount of receptor was determined by densitometric analysis and expressed as a percentage of control values. Only samples of biotinylated proteins lacking immunoreactivity to actin, which indicates contamination with intracellular proteins, were used for analyses. A , Representative Western blots of Kv7.2, Na-KATPase, and actin from total (T) and biotinylated (B) proteins. B , Quantitative analysis of the effect of gain and loss of NP1 on the surface/total levels of the Kv7.2 subunit of the voltage-gated Kv7/M K + channels. Values are mean ± SEM of shRdm ( n = 11), shNP1 ( n = 11), and pWPI-NP1 ( n = 5), from at least three independent experiments; * p

Techniques Used: Expressing, Cell Culture, Western Blot

2) Product Images from "Neuronal Pentraxin 1 Negatively Regulates Excitatory Synapse Density and Synaptic Plasticity"

Article Title: Neuronal Pentraxin 1 Negatively Regulates Excitatory Synapse Density and Synaptic Plasticity

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.2548-14.2015

NP1 interacts and colocalizes with Kv7.2 at presynaptic terminals of excitatory synapses and axonal growth cones. A – D , Representative Western blots of immunoprecipitation eluates separated in 6% Tris-Glycine (for high molecular weight proteins) and 4–12% Bis-Tris gels (for low molecular weight proteins). TL, Total protein lysate; Syx, syntaxin. A , Both native Kv7.2 and syntaxin 1A, but not Kv7.3, coprecipitate with NP1 in total brain extracts. B , Both native Kv7.2 and NP1 coprecipitate with syntaxin in total brain extracts. C , D , Recombinant NP1 coprecipitates Kv7.2, but not syntaxin or Kv7.3, in 293T cells transfected with NP1, 5Myc-Kv7.2, 2HA-Kv7.3, and syntaxin 1A cDNAs. Kv7.2 and Kv7.3 were immunoprecipitated with antibodies against their respective Myc and HA tags. E , F , Immunofluorescence studies and confocal microscopy were performed in high-density ( E ) or low-density ( F ) isolated cortical neurons. E , Top, Confocal sections of 0.772 μm in the z -plane showing immunofluorescence of NP1, VGLUT1, Kv7.2, and negative control (omitting primary antibodies). Bottom, Colocalization (in white) of the excitatory presynaptic marker VGLUT1 (blue) with NP1 (green) and Kv7.2 (red) is shown in a single section with the corresponding orthogonal views of the stack of confocal sections. White arrows indicate sites of colocalization. F , NP1 (green) and KV7.2 (magenta) immunofluorescence and DIC images of an isolated cortical cultured neuron (1×) with its corresponding axonal growth cone highlighted in a white square box, shown in a confocal section of 0.772 μm in the z -plane at higher (5×) magnification. The negative control for primary antibodies is shown in another growth cone on the right. The image in the bottom is the merge of NP1 and Kv7.2 immunofluorescence images in the single confocal section obtained at 5× showing colocalization (white) of NP1 and Kv7.2 in the growth cone, with the corresponding orthogonal views from its respective stack of confocal sections. Images were acquired using restricted spectral emission wavelength ranges chosen to avoid crosstalk or bleed-through between the three different channels. Scale bar, 5 μm.
Figure Legend Snippet: NP1 interacts and colocalizes with Kv7.2 at presynaptic terminals of excitatory synapses and axonal growth cones. A – D , Representative Western blots of immunoprecipitation eluates separated in 6% Tris-Glycine (for high molecular weight proteins) and 4–12% Bis-Tris gels (for low molecular weight proteins). TL, Total protein lysate; Syx, syntaxin. A , Both native Kv7.2 and syntaxin 1A, but not Kv7.3, coprecipitate with NP1 in total brain extracts. B , Both native Kv7.2 and NP1 coprecipitate with syntaxin in total brain extracts. C , D , Recombinant NP1 coprecipitates Kv7.2, but not syntaxin or Kv7.3, in 293T cells transfected with NP1, 5Myc-Kv7.2, 2HA-Kv7.3, and syntaxin 1A cDNAs. Kv7.2 and Kv7.3 were immunoprecipitated with antibodies against their respective Myc and HA tags. E , F , Immunofluorescence studies and confocal microscopy were performed in high-density ( E ) or low-density ( F ) isolated cortical neurons. E , Top, Confocal sections of 0.772 μm in the z -plane showing immunofluorescence of NP1, VGLUT1, Kv7.2, and negative control (omitting primary antibodies). Bottom, Colocalization (in white) of the excitatory presynaptic marker VGLUT1 (blue) with NP1 (green) and Kv7.2 (red) is shown in a single section with the corresponding orthogonal views of the stack of confocal sections. White arrows indicate sites of colocalization. F , NP1 (green) and KV7.2 (magenta) immunofluorescence and DIC images of an isolated cortical cultured neuron (1×) with its corresponding axonal growth cone highlighted in a white square box, shown in a confocal section of 0.772 μm in the z -plane at higher (5×) magnification. The negative control for primary antibodies is shown in another growth cone on the right. The image in the bottom is the merge of NP1 and Kv7.2 immunofluorescence images in the single confocal section obtained at 5× showing colocalization (white) of NP1 and Kv7.2 in the growth cone, with the corresponding orthogonal views from its respective stack of confocal sections. Images were acquired using restricted spectral emission wavelength ranges chosen to avoid crosstalk or bleed-through between the three different channels. Scale bar, 5 μm.

Techniques Used: Western Blot, Immunoprecipitation, Molecular Weight, Recombinant, Transfection, Immunofluorescence, Confocal Microscopy, Isolation, Negative Control, Marker, Cell Culture

Knockdown of NP1 reduces Kv7.2 surface expression and M-current. A , B , The proportion of Kv7.2 at the neuronal surface was determined by biotinylation (30 min at 4°C) of all surface proteins in cultured cortical neurons. The ratio of the surface over the total amount of receptor was determined by densitometric analysis and expressed as a percentage of control values. Only samples of biotinylated proteins lacking immunoreactivity to actin, which indicates contamination with intracellular proteins, were used for analyses. A , Representative Western blots of Kv7.2, Na-KATPase, and actin from total (T) and biotinylated (B) proteins. B , Quantitative analysis of the effect of gain and loss of NP1 on the surface/total levels of the Kv7.2 subunit of the voltage-gated Kv7/M K + channels. Values are mean ± SEM of shRdm ( n = 11), shNP1 ( n = 11), and pWPI-NP1 ( n = 5), from at least three independent experiments; * p
Figure Legend Snippet: Knockdown of NP1 reduces Kv7.2 surface expression and M-current. A , B , The proportion of Kv7.2 at the neuronal surface was determined by biotinylation (30 min at 4°C) of all surface proteins in cultured cortical neurons. The ratio of the surface over the total amount of receptor was determined by densitometric analysis and expressed as a percentage of control values. Only samples of biotinylated proteins lacking immunoreactivity to actin, which indicates contamination with intracellular proteins, were used for analyses. A , Representative Western blots of Kv7.2, Na-KATPase, and actin from total (T) and biotinylated (B) proteins. B , Quantitative analysis of the effect of gain and loss of NP1 on the surface/total levels of the Kv7.2 subunit of the voltage-gated Kv7/M K + channels. Values are mean ± SEM of shRdm ( n = 11), shNP1 ( n = 11), and pWPI-NP1 ( n = 5), from at least three independent experiments; * p

Techniques Used: Expressing, Cell Culture, Western Blot

3) Product Images from "Neuronal Pentraxin 1 Negatively Regulates Excitatory Synapse Density and Synaptic Plasticity"

Article Title: Neuronal Pentraxin 1 Negatively Regulates Excitatory Synapse Density and Synaptic Plasticity

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.2548-14.2015

NP1 interacts and colocalizes with Kv7.2 at presynaptic terminals of excitatory synapses and axonal growth cones. A – D , Representative Western blots of immunoprecipitation eluates separated in 6% Tris-Glycine (for high molecular weight proteins) and 4–12% Bis-Tris gels (for low molecular weight proteins). TL, Total protein lysate; Syx, syntaxin. A , Both native Kv7.2 and syntaxin 1A, but not Kv7.3, coprecipitate with NP1 in total brain extracts. B , Both native Kv7.2 and NP1 coprecipitate with syntaxin in total brain extracts. C , D , Recombinant NP1 coprecipitates Kv7.2, but not syntaxin or Kv7.3, in 293T cells transfected with NP1, 5Myc-Kv7.2, 2HA-Kv7.3, and syntaxin 1A cDNAs. Kv7.2 and Kv7.3 were immunoprecipitated with antibodies against their respective Myc and HA tags. E , F , Immunofluorescence studies and confocal microscopy were performed in high-density ( E ) or low-density ( F ) isolated cortical neurons. E , Top, Confocal sections of 0.772 μm in the z -plane showing immunofluorescence of NP1, VGLUT1, Kv7.2, and negative control (omitting primary antibodies). Bottom, Colocalization (in white) of the excitatory presynaptic marker VGLUT1 (blue) with NP1 (green) and Kv7.2 (red) is shown in a single section with the corresponding orthogonal views of the stack of confocal sections. White arrows indicate sites of colocalization. F , NP1 (green) and KV7.2 (magenta) immunofluorescence and DIC images of an isolated cortical cultured neuron (1×) with its corresponding axonal growth cone highlighted in a white square box, shown in a confocal section of 0.772 μm in the z -plane at higher (5×) magnification. The negative control for primary antibodies is shown in another growth cone on the right. The image in the bottom is the merge of NP1 and Kv7.2 immunofluorescence images in the single confocal section obtained at 5× showing colocalization (white) of NP1 and Kv7.2 in the growth cone, with the corresponding orthogonal views from its respective stack of confocal sections. Images were acquired using restricted spectral emission wavelength ranges chosen to avoid crosstalk or bleed-through between the three different channels. Scale bar, 5 μm.
Figure Legend Snippet: NP1 interacts and colocalizes with Kv7.2 at presynaptic terminals of excitatory synapses and axonal growth cones. A – D , Representative Western blots of immunoprecipitation eluates separated in 6% Tris-Glycine (for high molecular weight proteins) and 4–12% Bis-Tris gels (for low molecular weight proteins). TL, Total protein lysate; Syx, syntaxin. A , Both native Kv7.2 and syntaxin 1A, but not Kv7.3, coprecipitate with NP1 in total brain extracts. B , Both native Kv7.2 and NP1 coprecipitate with syntaxin in total brain extracts. C , D , Recombinant NP1 coprecipitates Kv7.2, but not syntaxin or Kv7.3, in 293T cells transfected with NP1, 5Myc-Kv7.2, 2HA-Kv7.3, and syntaxin 1A cDNAs. Kv7.2 and Kv7.3 were immunoprecipitated with antibodies against their respective Myc and HA tags. E , F , Immunofluorescence studies and confocal microscopy were performed in high-density ( E ) or low-density ( F ) isolated cortical neurons. E , Top, Confocal sections of 0.772 μm in the z -plane showing immunofluorescence of NP1, VGLUT1, Kv7.2, and negative control (omitting primary antibodies). Bottom, Colocalization (in white) of the excitatory presynaptic marker VGLUT1 (blue) with NP1 (green) and Kv7.2 (red) is shown in a single section with the corresponding orthogonal views of the stack of confocal sections. White arrows indicate sites of colocalization. F , NP1 (green) and KV7.2 (magenta) immunofluorescence and DIC images of an isolated cortical cultured neuron (1×) with its corresponding axonal growth cone highlighted in a white square box, shown in a confocal section of 0.772 μm in the z -plane at higher (5×) magnification. The negative control for primary antibodies is shown in another growth cone on the right. The image in the bottom is the merge of NP1 and Kv7.2 immunofluorescence images in the single confocal section obtained at 5× showing colocalization (white) of NP1 and Kv7.2 in the growth cone, with the corresponding orthogonal views from its respective stack of confocal sections. Images were acquired using restricted spectral emission wavelength ranges chosen to avoid crosstalk or bleed-through between the three different channels. Scale bar, 5 μm.

Techniques Used: Western Blot, Immunoprecipitation, Molecular Weight, Recombinant, Transfection, Immunofluorescence, Confocal Microscopy, Isolation, Negative Control, Marker, Cell Culture

Knockdown of NP1 reduces Kv7.2 surface expression and M-current. A , B , The proportion of Kv7.2 at the neuronal surface was determined by biotinylation (30 min at 4°C) of all surface proteins in cultured cortical neurons. The ratio of the surface over the total amount of receptor was determined by densitometric analysis and expressed as a percentage of control values. Only samples of biotinylated proteins lacking immunoreactivity to actin, which indicates contamination with intracellular proteins, were used for analyses. A , Representative Western blots of Kv7.2, Na-KATPase, and actin from total (T) and biotinylated (B) proteins. B , Quantitative analysis of the effect of gain and loss of NP1 on the surface/total levels of the Kv7.2 subunit of the voltage-gated Kv7/M K + channels. Values are mean ± SEM of shRdm ( n = 11), shNP1 ( n = 11), and pWPI-NP1 ( n = 5), from at least three independent experiments; * p
Figure Legend Snippet: Knockdown of NP1 reduces Kv7.2 surface expression and M-current. A , B , The proportion of Kv7.2 at the neuronal surface was determined by biotinylation (30 min at 4°C) of all surface proteins in cultured cortical neurons. The ratio of the surface over the total amount of receptor was determined by densitometric analysis and expressed as a percentage of control values. Only samples of biotinylated proteins lacking immunoreactivity to actin, which indicates contamination with intracellular proteins, were used for analyses. A , Representative Western blots of Kv7.2, Na-KATPase, and actin from total (T) and biotinylated (B) proteins. B , Quantitative analysis of the effect of gain and loss of NP1 on the surface/total levels of the Kv7.2 subunit of the voltage-gated Kv7/M K + channels. Values are mean ± SEM of shRdm ( n = 11), shNP1 ( n = 11), and pWPI-NP1 ( n = 5), from at least three independent experiments; * p

Techniques Used: Expressing, Cell Culture, Western Blot

4) Product Images from "Neuronal Pentraxin 1 Negatively Regulates Excitatory Synapse Density and Synaptic Plasticity"

Article Title: Neuronal Pentraxin 1 Negatively Regulates Excitatory Synapse Density and Synaptic Plasticity

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.2548-14.2015

NP1 interacts and colocalizes with Kv7.2 at presynaptic terminals of excitatory synapses and axonal growth cones. A – D , Representative Western blots of immunoprecipitation eluates separated in 6% Tris-Glycine (for high molecular weight proteins) and 4–12% Bis-Tris gels (for low molecular weight proteins). TL, Total protein lysate; Syx, syntaxin. A , Both native Kv7.2 and syntaxin 1A, but not Kv7.3, coprecipitate with NP1 in total brain extracts. B , Both native Kv7.2 and NP1 coprecipitate with syntaxin in total brain extracts. C , D , Recombinant NP1 coprecipitates Kv7.2, but not syntaxin or Kv7.3, in 293T cells transfected with NP1, 5Myc-Kv7.2, 2HA-Kv7.3, and syntaxin 1A cDNAs. Kv7.2 and Kv7.3 were immunoprecipitated with antibodies against their respective Myc and HA tags. E , F , Immunofluorescence studies and confocal microscopy were performed in high-density ( E ) or low-density ( F ) isolated cortical neurons. E , Top, Confocal sections of 0.772 μm in the z -plane showing immunofluorescence of NP1, VGLUT1, Kv7.2, and negative control (omitting primary antibodies). Bottom, Colocalization (in white) of the excitatory presynaptic marker VGLUT1 (blue) with NP1 (green) and Kv7.2 (red) is shown in a single section with the corresponding orthogonal views of the stack of confocal sections. White arrows indicate sites of colocalization. F , NP1 (green) and KV7.2 (magenta) immunofluorescence and DIC images of an isolated cortical cultured neuron (1×) with its corresponding axonal growth cone highlighted in a white square box, shown in a confocal section of 0.772 μm in the z -plane at higher (5×) magnification. The negative control for primary antibodies is shown in another growth cone on the right. The image in the bottom is the merge of NP1 and Kv7.2 immunofluorescence images in the single confocal section obtained at 5× showing colocalization (white) of NP1 and Kv7.2 in the growth cone, with the corresponding orthogonal views from its respective stack of confocal sections. Images were acquired using restricted spectral emission wavelength ranges chosen to avoid crosstalk or bleed-through between the three different channels. Scale bar, 5 μm.
Figure Legend Snippet: NP1 interacts and colocalizes with Kv7.2 at presynaptic terminals of excitatory synapses and axonal growth cones. A – D , Representative Western blots of immunoprecipitation eluates separated in 6% Tris-Glycine (for high molecular weight proteins) and 4–12% Bis-Tris gels (for low molecular weight proteins). TL, Total protein lysate; Syx, syntaxin. A , Both native Kv7.2 and syntaxin 1A, but not Kv7.3, coprecipitate with NP1 in total brain extracts. B , Both native Kv7.2 and NP1 coprecipitate with syntaxin in total brain extracts. C , D , Recombinant NP1 coprecipitates Kv7.2, but not syntaxin or Kv7.3, in 293T cells transfected with NP1, 5Myc-Kv7.2, 2HA-Kv7.3, and syntaxin 1A cDNAs. Kv7.2 and Kv7.3 were immunoprecipitated with antibodies against their respective Myc and HA tags. E , F , Immunofluorescence studies and confocal microscopy were performed in high-density ( E ) or low-density ( F ) isolated cortical neurons. E , Top, Confocal sections of 0.772 μm in the z -plane showing immunofluorescence of NP1, VGLUT1, Kv7.2, and negative control (omitting primary antibodies). Bottom, Colocalization (in white) of the excitatory presynaptic marker VGLUT1 (blue) with NP1 (green) and Kv7.2 (red) is shown in a single section with the corresponding orthogonal views of the stack of confocal sections. White arrows indicate sites of colocalization. F , NP1 (green) and KV7.2 (magenta) immunofluorescence and DIC images of an isolated cortical cultured neuron (1×) with its corresponding axonal growth cone highlighted in a white square box, shown in a confocal section of 0.772 μm in the z -plane at higher (5×) magnification. The negative control for primary antibodies is shown in another growth cone on the right. The image in the bottom is the merge of NP1 and Kv7.2 immunofluorescence images in the single confocal section obtained at 5× showing colocalization (white) of NP1 and Kv7.2 in the growth cone, with the corresponding orthogonal views from its respective stack of confocal sections. Images were acquired using restricted spectral emission wavelength ranges chosen to avoid crosstalk or bleed-through between the three different channels. Scale bar, 5 μm.

Techniques Used: Western Blot, Immunoprecipitation, Molecular Weight, Recombinant, Transfection, Immunofluorescence, Confocal Microscopy, Isolation, Negative Control, Marker, Cell Culture

Knockdown of NP1 reduces Kv7.2 surface expression and M-current. A , B , The proportion of Kv7.2 at the neuronal surface was determined by biotinylation (30 min at 4°C) of all surface proteins in cultured cortical neurons. The ratio of the surface over the total amount of receptor was determined by densitometric analysis and expressed as a percentage of control values. Only samples of biotinylated proteins lacking immunoreactivity to actin, which indicates contamination with intracellular proteins, were used for analyses. A , Representative Western blots of Kv7.2, Na-KATPase, and actin from total (T) and biotinylated (B) proteins. B , Quantitative analysis of the effect of gain and loss of NP1 on the surface/total levels of the Kv7.2 subunit of the voltage-gated Kv7/M K + channels. Values are mean ± SEM of shRdm ( n = 11), shNP1 ( n = 11), and pWPI-NP1 ( n = 5), from at least three independent experiments; * p
Figure Legend Snippet: Knockdown of NP1 reduces Kv7.2 surface expression and M-current. A , B , The proportion of Kv7.2 at the neuronal surface was determined by biotinylation (30 min at 4°C) of all surface proteins in cultured cortical neurons. The ratio of the surface over the total amount of receptor was determined by densitometric analysis and expressed as a percentage of control values. Only samples of biotinylated proteins lacking immunoreactivity to actin, which indicates contamination with intracellular proteins, were used for analyses. A , Representative Western blots of Kv7.2, Na-KATPase, and actin from total (T) and biotinylated (B) proteins. B , Quantitative analysis of the effect of gain and loss of NP1 on the surface/total levels of the Kv7.2 subunit of the voltage-gated Kv7/M K + channels. Values are mean ± SEM of shRdm ( n = 11), shNP1 ( n = 11), and pWPI-NP1 ( n = 5), from at least three independent experiments; * p

Techniques Used: Expressing, Cell Culture, Western Blot

5) Product Images from "Neuronal Pentraxin 1 Negatively Regulates Excitatory Synapse Density and Synaptic Plasticity"

Article Title: Neuronal Pentraxin 1 Negatively Regulates Excitatory Synapse Density and Synaptic Plasticity

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.2548-14.2015

NP1 interacts and colocalizes with Kv7.2 at presynaptic terminals of excitatory synapses and axonal growth cones. A – D , Representative Western blots of immunoprecipitation eluates separated in 6% Tris-Glycine (for high molecular weight proteins) and 4–12% Bis-Tris gels (for low molecular weight proteins). TL, Total protein lysate; Syx, syntaxin. A , Both native Kv7.2 and syntaxin 1A, but not Kv7.3, coprecipitate with NP1 in total brain extracts. B , Both native Kv7.2 and NP1 coprecipitate with syntaxin in total brain extracts. C , D , Recombinant NP1 coprecipitates Kv7.2, but not syntaxin or Kv7.3, in 293T cells transfected with NP1, 5Myc-Kv7.2, 2HA-Kv7.3, and syntaxin 1A cDNAs. Kv7.2 and Kv7.3 were immunoprecipitated with antibodies against their respective Myc and HA tags. E , F , Immunofluorescence studies and confocal microscopy were performed in high-density ( E ) or low-density ( F ) isolated cortical neurons. E , Top, Confocal sections of 0.772 μm in the z -plane showing immunofluorescence of NP1, VGLUT1, Kv7.2, and negative control (omitting primary antibodies). Bottom, Colocalization (in white) of the excitatory presynaptic marker VGLUT1 (blue) with NP1 (green) and Kv7.2 (red) is shown in a single section with the corresponding orthogonal views of the stack of confocal sections. White arrows indicate sites of colocalization. F , NP1 (green) and KV7.2 (magenta) immunofluorescence and DIC images of an isolated cortical cultured neuron (1×) with its corresponding axonal growth cone highlighted in a white square box, shown in a confocal section of 0.772 μm in the z -plane at higher (5×) magnification. The negative control for primary antibodies is shown in another growth cone on the right. The image in the bottom is the merge of NP1 and Kv7.2 immunofluorescence images in the single confocal section obtained at 5× showing colocalization (white) of NP1 and Kv7.2 in the growth cone, with the corresponding orthogonal views from its respective stack of confocal sections. Images were acquired using restricted spectral emission wavelength ranges chosen to avoid crosstalk or bleed-through between the three different channels. Scale bar, 5 μm.
Figure Legend Snippet: NP1 interacts and colocalizes with Kv7.2 at presynaptic terminals of excitatory synapses and axonal growth cones. A – D , Representative Western blots of immunoprecipitation eluates separated in 6% Tris-Glycine (for high molecular weight proteins) and 4–12% Bis-Tris gels (for low molecular weight proteins). TL, Total protein lysate; Syx, syntaxin. A , Both native Kv7.2 and syntaxin 1A, but not Kv7.3, coprecipitate with NP1 in total brain extracts. B , Both native Kv7.2 and NP1 coprecipitate with syntaxin in total brain extracts. C , D , Recombinant NP1 coprecipitates Kv7.2, but not syntaxin or Kv7.3, in 293T cells transfected with NP1, 5Myc-Kv7.2, 2HA-Kv7.3, and syntaxin 1A cDNAs. Kv7.2 and Kv7.3 were immunoprecipitated with antibodies against their respective Myc and HA tags. E , F , Immunofluorescence studies and confocal microscopy were performed in high-density ( E ) or low-density ( F ) isolated cortical neurons. E , Top, Confocal sections of 0.772 μm in the z -plane showing immunofluorescence of NP1, VGLUT1, Kv7.2, and negative control (omitting primary antibodies). Bottom, Colocalization (in white) of the excitatory presynaptic marker VGLUT1 (blue) with NP1 (green) and Kv7.2 (red) is shown in a single section with the corresponding orthogonal views of the stack of confocal sections. White arrows indicate sites of colocalization. F , NP1 (green) and KV7.2 (magenta) immunofluorescence and DIC images of an isolated cortical cultured neuron (1×) with its corresponding axonal growth cone highlighted in a white square box, shown in a confocal section of 0.772 μm in the z -plane at higher (5×) magnification. The negative control for primary antibodies is shown in another growth cone on the right. The image in the bottom is the merge of NP1 and Kv7.2 immunofluorescence images in the single confocal section obtained at 5× showing colocalization (white) of NP1 and Kv7.2 in the growth cone, with the corresponding orthogonal views from its respective stack of confocal sections. Images were acquired using restricted spectral emission wavelength ranges chosen to avoid crosstalk or bleed-through between the three different channels. Scale bar, 5 μm.

Techniques Used: Western Blot, Immunoprecipitation, Molecular Weight, Recombinant, Transfection, Immunofluorescence, Confocal Microscopy, Isolation, Negative Control, Marker, Cell Culture

Knockdown of NP1 reduces Kv7.2 surface expression and M-current. A , B , The proportion of Kv7.2 at the neuronal surface was determined by biotinylation (30 min at 4°C) of all surface proteins in cultured cortical neurons. The ratio of the surface over the total amount of receptor was determined by densitometric analysis and expressed as a percentage of control values. Only samples of biotinylated proteins lacking immunoreactivity to actin, which indicates contamination with intracellular proteins, were used for analyses. A , Representative Western blots of Kv7.2, Na-KATPase, and actin from total (T) and biotinylated (B) proteins. B , Quantitative analysis of the effect of gain and loss of NP1 on the surface/total levels of the Kv7.2 subunit of the voltage-gated Kv7/M K + channels. Values are mean ± SEM of shRdm ( n = 11), shNP1 ( n = 11), and pWPI-NP1 ( n = 5), from at least three independent experiments; * p
Figure Legend Snippet: Knockdown of NP1 reduces Kv7.2 surface expression and M-current. A , B , The proportion of Kv7.2 at the neuronal surface was determined by biotinylation (30 min at 4°C) of all surface proteins in cultured cortical neurons. The ratio of the surface over the total amount of receptor was determined by densitometric analysis and expressed as a percentage of control values. Only samples of biotinylated proteins lacking immunoreactivity to actin, which indicates contamination with intracellular proteins, were used for analyses. A , Representative Western blots of Kv7.2, Na-KATPase, and actin from total (T) and biotinylated (B) proteins. B , Quantitative analysis of the effect of gain and loss of NP1 on the surface/total levels of the Kv7.2 subunit of the voltage-gated Kv7/M K + channels. Values are mean ± SEM of shRdm ( n = 11), shNP1 ( n = 11), and pWPI-NP1 ( n = 5), from at least three independent experiments; * p

Techniques Used: Expressing, Cell Culture, Western Blot

6) Product Images from "Neuronal Pentraxin 1 Negatively Regulates Excitatory Synapse Density and Synaptic Plasticity"

Article Title: Neuronal Pentraxin 1 Negatively Regulates Excitatory Synapse Density and Synaptic Plasticity

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.2548-14.2015

NP1 interacts and colocalizes with Kv7.2 at presynaptic terminals of excitatory synapses and axonal growth cones. A – D , Representative Western blots of immunoprecipitation eluates separated in 6% Tris-Glycine (for high molecular weight proteins) and 4–12% Bis-Tris gels (for low molecular weight proteins). TL, Total protein lysate; Syx, syntaxin. A , Both native Kv7.2 and syntaxin 1A, but not Kv7.3, coprecipitate with NP1 in total brain extracts. B , Both native Kv7.2 and NP1 coprecipitate with syntaxin in total brain extracts. C , D , Recombinant NP1 coprecipitates Kv7.2, but not syntaxin or Kv7.3, in 293T cells transfected with NP1, 5Myc-Kv7.2, 2HA-Kv7.3, and syntaxin 1A cDNAs. Kv7.2 and Kv7.3 were immunoprecipitated with antibodies against their respective Myc and HA tags. E , F , Immunofluorescence studies and confocal microscopy were performed in high-density ( E ) or low-density ( F ) isolated cortical neurons. E , Top, Confocal sections of 0.772 μm in the z -plane showing immunofluorescence of NP1, VGLUT1, Kv7.2, and negative control (omitting primary antibodies). Bottom, Colocalization (in white) of the excitatory presynaptic marker VGLUT1 (blue) with NP1 (green) and Kv7.2 (red) is shown in a single section with the corresponding orthogonal views of the stack of confocal sections. White arrows indicate sites of colocalization. F , NP1 (green) and KV7.2 (magenta) immunofluorescence and DIC images of an isolated cortical cultured neuron (1×) with its corresponding axonal growth cone highlighted in a white square box, shown in a confocal section of 0.772 μm in the z -plane at higher (5×) magnification. The negative control for primary antibodies is shown in another growth cone on the right. The image in the bottom is the merge of NP1 and Kv7.2 immunofluorescence images in the single confocal section obtained at 5× showing colocalization (white) of NP1 and Kv7.2 in the growth cone, with the corresponding orthogonal views from its respective stack of confocal sections. Images were acquired using restricted spectral emission wavelength ranges chosen to avoid crosstalk or bleed-through between the three different channels. Scale bar, 5 μm.
Figure Legend Snippet: NP1 interacts and colocalizes with Kv7.2 at presynaptic terminals of excitatory synapses and axonal growth cones. A – D , Representative Western blots of immunoprecipitation eluates separated in 6% Tris-Glycine (for high molecular weight proteins) and 4–12% Bis-Tris gels (for low molecular weight proteins). TL, Total protein lysate; Syx, syntaxin. A , Both native Kv7.2 and syntaxin 1A, but not Kv7.3, coprecipitate with NP1 in total brain extracts. B , Both native Kv7.2 and NP1 coprecipitate with syntaxin in total brain extracts. C , D , Recombinant NP1 coprecipitates Kv7.2, but not syntaxin or Kv7.3, in 293T cells transfected with NP1, 5Myc-Kv7.2, 2HA-Kv7.3, and syntaxin 1A cDNAs. Kv7.2 and Kv7.3 were immunoprecipitated with antibodies against their respective Myc and HA tags. E , F , Immunofluorescence studies and confocal microscopy were performed in high-density ( E ) or low-density ( F ) isolated cortical neurons. E , Top, Confocal sections of 0.772 μm in the z -plane showing immunofluorescence of NP1, VGLUT1, Kv7.2, and negative control (omitting primary antibodies). Bottom, Colocalization (in white) of the excitatory presynaptic marker VGLUT1 (blue) with NP1 (green) and Kv7.2 (red) is shown in a single section with the corresponding orthogonal views of the stack of confocal sections. White arrows indicate sites of colocalization. F , NP1 (green) and KV7.2 (magenta) immunofluorescence and DIC images of an isolated cortical cultured neuron (1×) with its corresponding axonal growth cone highlighted in a white square box, shown in a confocal section of 0.772 μm in the z -plane at higher (5×) magnification. The negative control for primary antibodies is shown in another growth cone on the right. The image in the bottom is the merge of NP1 and Kv7.2 immunofluorescence images in the single confocal section obtained at 5× showing colocalization (white) of NP1 and Kv7.2 in the growth cone, with the corresponding orthogonal views from its respective stack of confocal sections. Images were acquired using restricted spectral emission wavelength ranges chosen to avoid crosstalk or bleed-through between the three different channels. Scale bar, 5 μm.

Techniques Used: Western Blot, Immunoprecipitation, Molecular Weight, Recombinant, Transfection, Immunofluorescence, Confocal Microscopy, Isolation, Negative Control, Marker, Cell Culture

Knockdown of NP1 reduces Kv7.2 surface expression and M-current. A , B , The proportion of Kv7.2 at the neuronal surface was determined by biotinylation (30 min at 4°C) of all surface proteins in cultured cortical neurons. The ratio of the surface over the total amount of receptor was determined by densitometric analysis and expressed as a percentage of control values. Only samples of biotinylated proteins lacking immunoreactivity to actin, which indicates contamination with intracellular proteins, were used for analyses. A , Representative Western blots of Kv7.2, Na-KATPase, and actin from total (T) and biotinylated (B) proteins. B , Quantitative analysis of the effect of gain and loss of NP1 on the surface/total levels of the Kv7.2 subunit of the voltage-gated Kv7/M K + channels. Values are mean ± SEM of shRdm ( n = 11), shNP1 ( n = 11), and pWPI-NP1 ( n = 5), from at least three independent experiments; * p
Figure Legend Snippet: Knockdown of NP1 reduces Kv7.2 surface expression and M-current. A , B , The proportion of Kv7.2 at the neuronal surface was determined by biotinylation (30 min at 4°C) of all surface proteins in cultured cortical neurons. The ratio of the surface over the total amount of receptor was determined by densitometric analysis and expressed as a percentage of control values. Only samples of biotinylated proteins lacking immunoreactivity to actin, which indicates contamination with intracellular proteins, were used for analyses. A , Representative Western blots of Kv7.2, Na-KATPase, and actin from total (T) and biotinylated (B) proteins. B , Quantitative analysis of the effect of gain and loss of NP1 on the surface/total levels of the Kv7.2 subunit of the voltage-gated Kv7/M K + channels. Values are mean ± SEM of shRdm ( n = 11), shNP1 ( n = 11), and pWPI-NP1 ( n = 5), from at least three independent experiments; * p

Techniques Used: Expressing, Cell Culture, Western Blot

7) Product Images from "Neuronal Pentraxin 1 Negatively Regulates Excitatory Synapse Density and Synaptic Plasticity"

Article Title: Neuronal Pentraxin 1 Negatively Regulates Excitatory Synapse Density and Synaptic Plasticity

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.2548-14.2015

NP1 interacts and colocalizes with Kv7.2 at presynaptic terminals of excitatory synapses and axonal growth cones. A – D , Representative Western blots of immunoprecipitation eluates separated in 6% Tris-Glycine (for high molecular weight proteins) and 4–12% Bis-Tris gels (for low molecular weight proteins). TL, Total protein lysate; Syx, syntaxin. A , Both native Kv7.2 and syntaxin 1A, but not Kv7.3, coprecipitate with NP1 in total brain extracts. B , Both native Kv7.2 and NP1 coprecipitate with syntaxin in total brain extracts. C , D , Recombinant NP1 coprecipitates Kv7.2, but not syntaxin or Kv7.3, in 293T cells transfected with NP1, 5Myc-Kv7.2, 2HA-Kv7.3, and syntaxin 1A cDNAs. Kv7.2 and Kv7.3 were immunoprecipitated with antibodies against their respective Myc and HA tags. E , F , Immunofluorescence studies and confocal microscopy were performed in high-density ( E ) or low-density ( F ) isolated cortical neurons. E , Top, Confocal sections of 0.772 μm in the z -plane showing immunofluorescence of NP1, VGLUT1, Kv7.2, and negative control (omitting primary antibodies). Bottom, Colocalization (in white) of the excitatory presynaptic marker VGLUT1 (blue) with NP1 (green) and Kv7.2 (red) is shown in a single section with the corresponding orthogonal views of the stack of confocal sections. White arrows indicate sites of colocalization. F , NP1 (green) and KV7.2 (magenta) immunofluorescence and DIC images of an isolated cortical cultured neuron (1×) with its corresponding axonal growth cone highlighted in a white square box, shown in a confocal section of 0.772 μm in the z -plane at higher (5×) magnification. The negative control for primary antibodies is shown in another growth cone on the right. The image in the bottom is the merge of NP1 and Kv7.2 immunofluorescence images in the single confocal section obtained at 5× showing colocalization (white) of NP1 and Kv7.2 in the growth cone, with the corresponding orthogonal views from its respective stack of confocal sections. Images were acquired using restricted spectral emission wavelength ranges chosen to avoid crosstalk or bleed-through between the three different channels. Scale bar, 5 μm.
Figure Legend Snippet: NP1 interacts and colocalizes with Kv7.2 at presynaptic terminals of excitatory synapses and axonal growth cones. A – D , Representative Western blots of immunoprecipitation eluates separated in 6% Tris-Glycine (for high molecular weight proteins) and 4–12% Bis-Tris gels (for low molecular weight proteins). TL, Total protein lysate; Syx, syntaxin. A , Both native Kv7.2 and syntaxin 1A, but not Kv7.3, coprecipitate with NP1 in total brain extracts. B , Both native Kv7.2 and NP1 coprecipitate with syntaxin in total brain extracts. C , D , Recombinant NP1 coprecipitates Kv7.2, but not syntaxin or Kv7.3, in 293T cells transfected with NP1, 5Myc-Kv7.2, 2HA-Kv7.3, and syntaxin 1A cDNAs. Kv7.2 and Kv7.3 were immunoprecipitated with antibodies against their respective Myc and HA tags. E , F , Immunofluorescence studies and confocal microscopy were performed in high-density ( E ) or low-density ( F ) isolated cortical neurons. E , Top, Confocal sections of 0.772 μm in the z -plane showing immunofluorescence of NP1, VGLUT1, Kv7.2, and negative control (omitting primary antibodies). Bottom, Colocalization (in white) of the excitatory presynaptic marker VGLUT1 (blue) with NP1 (green) and Kv7.2 (red) is shown in a single section with the corresponding orthogonal views of the stack of confocal sections. White arrows indicate sites of colocalization. F , NP1 (green) and KV7.2 (magenta) immunofluorescence and DIC images of an isolated cortical cultured neuron (1×) with its corresponding axonal growth cone highlighted in a white square box, shown in a confocal section of 0.772 μm in the z -plane at higher (5×) magnification. The negative control for primary antibodies is shown in another growth cone on the right. The image in the bottom is the merge of NP1 and Kv7.2 immunofluorescence images in the single confocal section obtained at 5× showing colocalization (white) of NP1 and Kv7.2 in the growth cone, with the corresponding orthogonal views from its respective stack of confocal sections. Images were acquired using restricted spectral emission wavelength ranges chosen to avoid crosstalk or bleed-through between the three different channels. Scale bar, 5 μm.

Techniques Used: Western Blot, Immunoprecipitation, Molecular Weight, Recombinant, Transfection, Immunofluorescence, Confocal Microscopy, Isolation, Negative Control, Marker, Cell Culture

Knockdown of NP1 reduces Kv7.2 surface expression and M-current. A , B , The proportion of Kv7.2 at the neuronal surface was determined by biotinylation (30 min at 4°C) of all surface proteins in cultured cortical neurons. The ratio of the surface over the total amount of receptor was determined by densitometric analysis and expressed as a percentage of control values. Only samples of biotinylated proteins lacking immunoreactivity to actin, which indicates contamination with intracellular proteins, were used for analyses. A , Representative Western blots of Kv7.2, Na-KATPase, and actin from total (T) and biotinylated (B) proteins. B , Quantitative analysis of the effect of gain and loss of NP1 on the surface/total levels of the Kv7.2 subunit of the voltage-gated Kv7/M K + channels. Values are mean ± SEM of shRdm ( n = 11), shNP1 ( n = 11), and pWPI-NP1 ( n = 5), from at least three independent experiments; * p
Figure Legend Snippet: Knockdown of NP1 reduces Kv7.2 surface expression and M-current. A , B , The proportion of Kv7.2 at the neuronal surface was determined by biotinylation (30 min at 4°C) of all surface proteins in cultured cortical neurons. The ratio of the surface over the total amount of receptor was determined by densitometric analysis and expressed as a percentage of control values. Only samples of biotinylated proteins lacking immunoreactivity to actin, which indicates contamination with intracellular proteins, were used for analyses. A , Representative Western blots of Kv7.2, Na-KATPase, and actin from total (T) and biotinylated (B) proteins. B , Quantitative analysis of the effect of gain and loss of NP1 on the surface/total levels of the Kv7.2 subunit of the voltage-gated Kv7/M K + channels. Values are mean ± SEM of shRdm ( n = 11), shNP1 ( n = 11), and pWPI-NP1 ( n = 5), from at least three independent experiments; * p

Techniques Used: Expressing, Cell Culture, Western Blot

8) Product Images from "Neuronal Pentraxin 1 Negatively Regulates Excitatory Synapse Density and Synaptic Plasticity"

Article Title: Neuronal Pentraxin 1 Negatively Regulates Excitatory Synapse Density and Synaptic Plasticity

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.2548-14.2015

NP1 interacts and colocalizes with Kv7.2 at presynaptic terminals of excitatory synapses and axonal growth cones. A – D , Representative Western blots of immunoprecipitation eluates separated in 6% Tris-Glycine (for high molecular weight proteins) and 4–12% Bis-Tris gels (for low molecular weight proteins). TL, Total protein lysate; Syx, syntaxin. A , Both native Kv7.2 and syntaxin 1A, but not Kv7.3, coprecipitate with NP1 in total brain extracts. B , Both native Kv7.2 and NP1 coprecipitate with syntaxin in total brain extracts. C , D , Recombinant NP1 coprecipitates Kv7.2, but not syntaxin or Kv7.3, in 293T cells transfected with NP1, 5Myc-Kv7.2, 2HA-Kv7.3, and syntaxin 1A cDNAs. Kv7.2 and Kv7.3 were immunoprecipitated with antibodies against their respective Myc and HA tags. E , F , Immunofluorescence studies and confocal microscopy were performed in high-density ( E ) or low-density ( F ) isolated cortical neurons. E , Top, Confocal sections of 0.772 μm in the z -plane showing immunofluorescence of NP1, VGLUT1, Kv7.2, and negative control (omitting primary antibodies). Bottom, Colocalization (in white) of the excitatory presynaptic marker VGLUT1 (blue) with NP1 (green) and Kv7.2 (red) is shown in a single section with the corresponding orthogonal views of the stack of confocal sections. White arrows indicate sites of colocalization. F , NP1 (green) and KV7.2 (magenta) immunofluorescence and DIC images of an isolated cortical cultured neuron (1×) with its corresponding axonal growth cone highlighted in a white square box, shown in a confocal section of 0.772 μm in the z -plane at higher (5×) magnification. The negative control for primary antibodies is shown in another growth cone on the right. The image in the bottom is the merge of NP1 and Kv7.2 immunofluorescence images in the single confocal section obtained at 5× showing colocalization (white) of NP1 and Kv7.2 in the growth cone, with the corresponding orthogonal views from its respective stack of confocal sections. Images were acquired using restricted spectral emission wavelength ranges chosen to avoid crosstalk or bleed-through between the three different channels. Scale bar, 5 μm.
Figure Legend Snippet: NP1 interacts and colocalizes with Kv7.2 at presynaptic terminals of excitatory synapses and axonal growth cones. A – D , Representative Western blots of immunoprecipitation eluates separated in 6% Tris-Glycine (for high molecular weight proteins) and 4–12% Bis-Tris gels (for low molecular weight proteins). TL, Total protein lysate; Syx, syntaxin. A , Both native Kv7.2 and syntaxin 1A, but not Kv7.3, coprecipitate with NP1 in total brain extracts. B , Both native Kv7.2 and NP1 coprecipitate with syntaxin in total brain extracts. C , D , Recombinant NP1 coprecipitates Kv7.2, but not syntaxin or Kv7.3, in 293T cells transfected with NP1, 5Myc-Kv7.2, 2HA-Kv7.3, and syntaxin 1A cDNAs. Kv7.2 and Kv7.3 were immunoprecipitated with antibodies against their respective Myc and HA tags. E , F , Immunofluorescence studies and confocal microscopy were performed in high-density ( E ) or low-density ( F ) isolated cortical neurons. E , Top, Confocal sections of 0.772 μm in the z -plane showing immunofluorescence of NP1, VGLUT1, Kv7.2, and negative control (omitting primary antibodies). Bottom, Colocalization (in white) of the excitatory presynaptic marker VGLUT1 (blue) with NP1 (green) and Kv7.2 (red) is shown in a single section with the corresponding orthogonal views of the stack of confocal sections. White arrows indicate sites of colocalization. F , NP1 (green) and KV7.2 (magenta) immunofluorescence and DIC images of an isolated cortical cultured neuron (1×) with its corresponding axonal growth cone highlighted in a white square box, shown in a confocal section of 0.772 μm in the z -plane at higher (5×) magnification. The negative control for primary antibodies is shown in another growth cone on the right. The image in the bottom is the merge of NP1 and Kv7.2 immunofluorescence images in the single confocal section obtained at 5× showing colocalization (white) of NP1 and Kv7.2 in the growth cone, with the corresponding orthogonal views from its respective stack of confocal sections. Images were acquired using restricted spectral emission wavelength ranges chosen to avoid crosstalk or bleed-through between the three different channels. Scale bar, 5 μm.

Techniques Used: Western Blot, Immunoprecipitation, Molecular Weight, Recombinant, Transfection, Immunofluorescence, Confocal Microscopy, Isolation, Negative Control, Marker, Cell Culture

Knockdown of NP1 reduces Kv7.2 surface expression and M-current. A , B , The proportion of Kv7.2 at the neuronal surface was determined by biotinylation (30 min at 4°C) of all surface proteins in cultured cortical neurons. The ratio of the surface over the total amount of receptor was determined by densitometric analysis and expressed as a percentage of control values. Only samples of biotinylated proteins lacking immunoreactivity to actin, which indicates contamination with intracellular proteins, were used for analyses. A , Representative Western blots of Kv7.2, Na-KATPase, and actin from total (T) and biotinylated (B) proteins. B , Quantitative analysis of the effect of gain and loss of NP1 on the surface/total levels of the Kv7.2 subunit of the voltage-gated Kv7/M K + channels. Values are mean ± SEM of shRdm ( n = 11), shNP1 ( n = 11), and pWPI-NP1 ( n = 5), from at least three independent experiments; * p
Figure Legend Snippet: Knockdown of NP1 reduces Kv7.2 surface expression and M-current. A , B , The proportion of Kv7.2 at the neuronal surface was determined by biotinylation (30 min at 4°C) of all surface proteins in cultured cortical neurons. The ratio of the surface over the total amount of receptor was determined by densitometric analysis and expressed as a percentage of control values. Only samples of biotinylated proteins lacking immunoreactivity to actin, which indicates contamination with intracellular proteins, were used for analyses. A , Representative Western blots of Kv7.2, Na-KATPase, and actin from total (T) and biotinylated (B) proteins. B , Quantitative analysis of the effect of gain and loss of NP1 on the surface/total levels of the Kv7.2 subunit of the voltage-gated Kv7/M K + channels. Values are mean ± SEM of shRdm ( n = 11), shNP1 ( n = 11), and pWPI-NP1 ( n = 5), from at least three independent experiments; * p

Techniques Used: Expressing, Cell Culture, Western Blot

9) Product Images from "Neuronal Pentraxin 1 Negatively Regulates Excitatory Synapse Density and Synaptic Plasticity"

Article Title: Neuronal Pentraxin 1 Negatively Regulates Excitatory Synapse Density and Synaptic Plasticity

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.2548-14.2015

NP1 interacts and colocalizes with Kv7.2 at presynaptic terminals of excitatory synapses and axonal growth cones. A – D , Representative Western blots of immunoprecipitation eluates separated in 6% Tris-Glycine (for high molecular weight proteins) and 4–12% Bis-Tris gels (for low molecular weight proteins). TL, Total protein lysate; Syx, syntaxin. A , Both native Kv7.2 and syntaxin 1A, but not Kv7.3, coprecipitate with NP1 in total brain extracts. B , Both native Kv7.2 and NP1 coprecipitate with syntaxin in total brain extracts. C , D , Recombinant NP1 coprecipitates Kv7.2, but not syntaxin or Kv7.3, in 293T cells transfected with NP1, 5Myc-Kv7.2, 2HA-Kv7.3, and syntaxin 1A cDNAs. Kv7.2 and Kv7.3 were immunoprecipitated with antibodies against their respective Myc and HA tags. E , F , Immunofluorescence studies and confocal microscopy were performed in high-density ( E ) or low-density ( F ) isolated cortical neurons. E , Top, Confocal sections of 0.772 μm in the z -plane showing immunofluorescence of NP1, VGLUT1, Kv7.2, and negative control (omitting primary antibodies). Bottom, Colocalization (in white) of the excitatory presynaptic marker VGLUT1 (blue) with NP1 (green) and Kv7.2 (red) is shown in a single section with the corresponding orthogonal views of the stack of confocal sections. White arrows indicate sites of colocalization. F , NP1 (green) and KV7.2 (magenta) immunofluorescence and DIC images of an isolated cortical cultured neuron (1×) with its corresponding axonal growth cone highlighted in a white square box, shown in a confocal section of 0.772 μm in the z -plane at higher (5×) magnification. The negative control for primary antibodies is shown in another growth cone on the right. The image in the bottom is the merge of NP1 and Kv7.2 immunofluorescence images in the single confocal section obtained at 5× showing colocalization (white) of NP1 and Kv7.2 in the growth cone, with the corresponding orthogonal views from its respective stack of confocal sections. Images were acquired using restricted spectral emission wavelength ranges chosen to avoid crosstalk or bleed-through between the three different channels. Scale bar, 5 μm.
Figure Legend Snippet: NP1 interacts and colocalizes with Kv7.2 at presynaptic terminals of excitatory synapses and axonal growth cones. A – D , Representative Western blots of immunoprecipitation eluates separated in 6% Tris-Glycine (for high molecular weight proteins) and 4–12% Bis-Tris gels (for low molecular weight proteins). TL, Total protein lysate; Syx, syntaxin. A , Both native Kv7.2 and syntaxin 1A, but not Kv7.3, coprecipitate with NP1 in total brain extracts. B , Both native Kv7.2 and NP1 coprecipitate with syntaxin in total brain extracts. C , D , Recombinant NP1 coprecipitates Kv7.2, but not syntaxin or Kv7.3, in 293T cells transfected with NP1, 5Myc-Kv7.2, 2HA-Kv7.3, and syntaxin 1A cDNAs. Kv7.2 and Kv7.3 were immunoprecipitated with antibodies against their respective Myc and HA tags. E , F , Immunofluorescence studies and confocal microscopy were performed in high-density ( E ) or low-density ( F ) isolated cortical neurons. E , Top, Confocal sections of 0.772 μm in the z -plane showing immunofluorescence of NP1, VGLUT1, Kv7.2, and negative control (omitting primary antibodies). Bottom, Colocalization (in white) of the excitatory presynaptic marker VGLUT1 (blue) with NP1 (green) and Kv7.2 (red) is shown in a single section with the corresponding orthogonal views of the stack of confocal sections. White arrows indicate sites of colocalization. F , NP1 (green) and KV7.2 (magenta) immunofluorescence and DIC images of an isolated cortical cultured neuron (1×) with its corresponding axonal growth cone highlighted in a white square box, shown in a confocal section of 0.772 μm in the z -plane at higher (5×) magnification. The negative control for primary antibodies is shown in another growth cone on the right. The image in the bottom is the merge of NP1 and Kv7.2 immunofluorescence images in the single confocal section obtained at 5× showing colocalization (white) of NP1 and Kv7.2 in the growth cone, with the corresponding orthogonal views from its respective stack of confocal sections. Images were acquired using restricted spectral emission wavelength ranges chosen to avoid crosstalk or bleed-through between the three different channels. Scale bar, 5 μm.

Techniques Used: Western Blot, Immunoprecipitation, Molecular Weight, Recombinant, Transfection, Immunofluorescence, Confocal Microscopy, Isolation, Negative Control, Marker, Cell Culture

Knockdown of NP1 reduces Kv7.2 surface expression and M-current. A , B , The proportion of Kv7.2 at the neuronal surface was determined by biotinylation (30 min at 4°C) of all surface proteins in cultured cortical neurons. The ratio of the surface over the total amount of receptor was determined by densitometric analysis and expressed as a percentage of control values. Only samples of biotinylated proteins lacking immunoreactivity to actin, which indicates contamination with intracellular proteins, were used for analyses. A , Representative Western blots of Kv7.2, Na-KATPase, and actin from total (T) and biotinylated (B) proteins. B , Quantitative analysis of the effect of gain and loss of NP1 on the surface/total levels of the Kv7.2 subunit of the voltage-gated Kv7/M K + channels. Values are mean ± SEM of shRdm ( n = 11), shNP1 ( n = 11), and pWPI-NP1 ( n = 5), from at least three independent experiments; * p
Figure Legend Snippet: Knockdown of NP1 reduces Kv7.2 surface expression and M-current. A , B , The proportion of Kv7.2 at the neuronal surface was determined by biotinylation (30 min at 4°C) of all surface proteins in cultured cortical neurons. The ratio of the surface over the total amount of receptor was determined by densitometric analysis and expressed as a percentage of control values. Only samples of biotinylated proteins lacking immunoreactivity to actin, which indicates contamination with intracellular proteins, were used for analyses. A , Representative Western blots of Kv7.2, Na-KATPase, and actin from total (T) and biotinylated (B) proteins. B , Quantitative analysis of the effect of gain and loss of NP1 on the surface/total levels of the Kv7.2 subunit of the voltage-gated Kv7/M K + channels. Values are mean ± SEM of shRdm ( n = 11), shNP1 ( n = 11), and pWPI-NP1 ( n = 5), from at least three independent experiments; * p

Techniques Used: Expressing, Cell Culture, Western Blot

10) Product Images from "Neuronal Pentraxin 1 Negatively Regulates Excitatory Synapse Density and Synaptic Plasticity"

Article Title: Neuronal Pentraxin 1 Negatively Regulates Excitatory Synapse Density and Synaptic Plasticity

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.2548-14.2015

NP1 interacts and colocalizes with Kv7.2 at presynaptic terminals of excitatory synapses and axonal growth cones. A – D , Representative Western blots of immunoprecipitation eluates separated in 6% Tris-Glycine (for high molecular weight proteins) and 4–12% Bis-Tris gels (for low molecular weight proteins). TL, Total protein lysate; Syx, syntaxin. A , Both native Kv7.2 and syntaxin 1A, but not Kv7.3, coprecipitate with NP1 in total brain extracts. B , Both native Kv7.2 and NP1 coprecipitate with syntaxin in total brain extracts. C , D , Recombinant NP1 coprecipitates Kv7.2, but not syntaxin or Kv7.3, in 293T cells transfected with NP1, 5Myc-Kv7.2, 2HA-Kv7.3, and syntaxin 1A cDNAs. Kv7.2 and Kv7.3 were immunoprecipitated with antibodies against their respective Myc and HA tags. E , F , Immunofluorescence studies and confocal microscopy were performed in high-density ( E ) or low-density ( F ) isolated cortical neurons. E , Top, Confocal sections of 0.772 μm in the z -plane showing immunofluorescence of NP1, VGLUT1, Kv7.2, and negative control (omitting primary antibodies). Bottom, Colocalization (in white) of the excitatory presynaptic marker VGLUT1 (blue) with NP1 (green) and Kv7.2 (red) is shown in a single section with the corresponding orthogonal views of the stack of confocal sections. White arrows indicate sites of colocalization. F , NP1 (green) and KV7.2 (magenta) immunofluorescence and DIC images of an isolated cortical cultured neuron (1×) with its corresponding axonal growth cone highlighted in a white square box, shown in a confocal section of 0.772 μm in the z -plane at higher (5×) magnification. The negative control for primary antibodies is shown in another growth cone on the right. The image in the bottom is the merge of NP1 and Kv7.2 immunofluorescence images in the single confocal section obtained at 5× showing colocalization (white) of NP1 and Kv7.2 in the growth cone, with the corresponding orthogonal views from its respective stack of confocal sections. Images were acquired using restricted spectral emission wavelength ranges chosen to avoid crosstalk or bleed-through between the three different channels. Scale bar, 5 μm.
Figure Legend Snippet: NP1 interacts and colocalizes with Kv7.2 at presynaptic terminals of excitatory synapses and axonal growth cones. A – D , Representative Western blots of immunoprecipitation eluates separated in 6% Tris-Glycine (for high molecular weight proteins) and 4–12% Bis-Tris gels (for low molecular weight proteins). TL, Total protein lysate; Syx, syntaxin. A , Both native Kv7.2 and syntaxin 1A, but not Kv7.3, coprecipitate with NP1 in total brain extracts. B , Both native Kv7.2 and NP1 coprecipitate with syntaxin in total brain extracts. C , D , Recombinant NP1 coprecipitates Kv7.2, but not syntaxin or Kv7.3, in 293T cells transfected with NP1, 5Myc-Kv7.2, 2HA-Kv7.3, and syntaxin 1A cDNAs. Kv7.2 and Kv7.3 were immunoprecipitated with antibodies against their respective Myc and HA tags. E , F , Immunofluorescence studies and confocal microscopy were performed in high-density ( E ) or low-density ( F ) isolated cortical neurons. E , Top, Confocal sections of 0.772 μm in the z -plane showing immunofluorescence of NP1, VGLUT1, Kv7.2, and negative control (omitting primary antibodies). Bottom, Colocalization (in white) of the excitatory presynaptic marker VGLUT1 (blue) with NP1 (green) and Kv7.2 (red) is shown in a single section with the corresponding orthogonal views of the stack of confocal sections. White arrows indicate sites of colocalization. F , NP1 (green) and KV7.2 (magenta) immunofluorescence and DIC images of an isolated cortical cultured neuron (1×) with its corresponding axonal growth cone highlighted in a white square box, shown in a confocal section of 0.772 μm in the z -plane at higher (5×) magnification. The negative control for primary antibodies is shown in another growth cone on the right. The image in the bottom is the merge of NP1 and Kv7.2 immunofluorescence images in the single confocal section obtained at 5× showing colocalization (white) of NP1 and Kv7.2 in the growth cone, with the corresponding orthogonal views from its respective stack of confocal sections. Images were acquired using restricted spectral emission wavelength ranges chosen to avoid crosstalk or bleed-through between the three different channels. Scale bar, 5 μm.

Techniques Used: Western Blot, Immunoprecipitation, Molecular Weight, Recombinant, Transfection, Immunofluorescence, Confocal Microscopy, Isolation, Negative Control, Marker, Cell Culture

Knockdown of NP1 reduces Kv7.2 surface expression and M-current. A , B , The proportion of Kv7.2 at the neuronal surface was determined by biotinylation (30 min at 4°C) of all surface proteins in cultured cortical neurons. The ratio of the surface over the total amount of receptor was determined by densitometric analysis and expressed as a percentage of control values. Only samples of biotinylated proteins lacking immunoreactivity to actin, which indicates contamination with intracellular proteins, were used for analyses. A , Representative Western blots of Kv7.2, Na-KATPase, and actin from total (T) and biotinylated (B) proteins. B , Quantitative analysis of the effect of gain and loss of NP1 on the surface/total levels of the Kv7.2 subunit of the voltage-gated Kv7/M K + channels. Values are mean ± SEM of shRdm ( n = 11), shNP1 ( n = 11), and pWPI-NP1 ( n = 5), from at least three independent experiments; * p
Figure Legend Snippet: Knockdown of NP1 reduces Kv7.2 surface expression and M-current. A , B , The proportion of Kv7.2 at the neuronal surface was determined by biotinylation (30 min at 4°C) of all surface proteins in cultured cortical neurons. The ratio of the surface over the total amount of receptor was determined by densitometric analysis and expressed as a percentage of control values. Only samples of biotinylated proteins lacking immunoreactivity to actin, which indicates contamination with intracellular proteins, were used for analyses. A , Representative Western blots of Kv7.2, Na-KATPase, and actin from total (T) and biotinylated (B) proteins. B , Quantitative analysis of the effect of gain and loss of NP1 on the surface/total levels of the Kv7.2 subunit of the voltage-gated Kv7/M K + channels. Values are mean ± SEM of shRdm ( n = 11), shNP1 ( n = 11), and pWPI-NP1 ( n = 5), from at least three independent experiments; * p

Techniques Used: Expressing, Cell Culture, Western Blot

11) Product Images from "Neuronal Pentraxin 1 Negatively Regulates Excitatory Synapse Density and Synaptic Plasticity"

Article Title: Neuronal Pentraxin 1 Negatively Regulates Excitatory Synapse Density and Synaptic Plasticity

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.2548-14.2015

NP1 interacts and colocalizes with Kv7.2 at presynaptic terminals of excitatory synapses and axonal growth cones. A – D , Representative Western blots of immunoprecipitation eluates separated in 6% Tris-Glycine (for high molecular weight proteins) and 4–12% Bis-Tris gels (for low molecular weight proteins). TL, Total protein lysate; Syx, syntaxin. A , Both native Kv7.2 and syntaxin 1A, but not Kv7.3, coprecipitate with NP1 in total brain extracts. B , Both native Kv7.2 and NP1 coprecipitate with syntaxin in total brain extracts. C , D , Recombinant NP1 coprecipitates Kv7.2, but not syntaxin or Kv7.3, in 293T cells transfected with NP1, 5Myc-Kv7.2, 2HA-Kv7.3, and syntaxin 1A cDNAs. Kv7.2 and Kv7.3 were immunoprecipitated with antibodies against their respective Myc and HA tags. E , F , Immunofluorescence studies and confocal microscopy were performed in high-density ( E ) or low-density ( F ) isolated cortical neurons. E , Top, Confocal sections of 0.772 μm in the z -plane showing immunofluorescence of NP1, VGLUT1, Kv7.2, and negative control (omitting primary antibodies). Bottom, Colocalization (in white) of the excitatory presynaptic marker VGLUT1 (blue) with NP1 (green) and Kv7.2 (red) is shown in a single section with the corresponding orthogonal views of the stack of confocal sections. White arrows indicate sites of colocalization. F , NP1 (green) and KV7.2 (magenta) immunofluorescence and DIC images of an isolated cortical cultured neuron (1×) with its corresponding axonal growth cone highlighted in a white square box, shown in a confocal section of 0.772 μm in the z -plane at higher (5×) magnification. The negative control for primary antibodies is shown in another growth cone on the right. The image in the bottom is the merge of NP1 and Kv7.2 immunofluorescence images in the single confocal section obtained at 5× showing colocalization (white) of NP1 and Kv7.2 in the growth cone, with the corresponding orthogonal views from its respective stack of confocal sections. Images were acquired using restricted spectral emission wavelength ranges chosen to avoid crosstalk or bleed-through between the three different channels. Scale bar, 5 μm.
Figure Legend Snippet: NP1 interacts and colocalizes with Kv7.2 at presynaptic terminals of excitatory synapses and axonal growth cones. A – D , Representative Western blots of immunoprecipitation eluates separated in 6% Tris-Glycine (for high molecular weight proteins) and 4–12% Bis-Tris gels (for low molecular weight proteins). TL, Total protein lysate; Syx, syntaxin. A , Both native Kv7.2 and syntaxin 1A, but not Kv7.3, coprecipitate with NP1 in total brain extracts. B , Both native Kv7.2 and NP1 coprecipitate with syntaxin in total brain extracts. C , D , Recombinant NP1 coprecipitates Kv7.2, but not syntaxin or Kv7.3, in 293T cells transfected with NP1, 5Myc-Kv7.2, 2HA-Kv7.3, and syntaxin 1A cDNAs. Kv7.2 and Kv7.3 were immunoprecipitated with antibodies against their respective Myc and HA tags. E , F , Immunofluorescence studies and confocal microscopy were performed in high-density ( E ) or low-density ( F ) isolated cortical neurons. E , Top, Confocal sections of 0.772 μm in the z -plane showing immunofluorescence of NP1, VGLUT1, Kv7.2, and negative control (omitting primary antibodies). Bottom, Colocalization (in white) of the excitatory presynaptic marker VGLUT1 (blue) with NP1 (green) and Kv7.2 (red) is shown in a single section with the corresponding orthogonal views of the stack of confocal sections. White arrows indicate sites of colocalization. F , NP1 (green) and KV7.2 (magenta) immunofluorescence and DIC images of an isolated cortical cultured neuron (1×) with its corresponding axonal growth cone highlighted in a white square box, shown in a confocal section of 0.772 μm in the z -plane at higher (5×) magnification. The negative control for primary antibodies is shown in another growth cone on the right. The image in the bottom is the merge of NP1 and Kv7.2 immunofluorescence images in the single confocal section obtained at 5× showing colocalization (white) of NP1 and Kv7.2 in the growth cone, with the corresponding orthogonal views from its respective stack of confocal sections. Images were acquired using restricted spectral emission wavelength ranges chosen to avoid crosstalk or bleed-through between the three different channels. Scale bar, 5 μm.

Techniques Used: Western Blot, Immunoprecipitation, Molecular Weight, Recombinant, Transfection, Immunofluorescence, Confocal Microscopy, Isolation, Negative Control, Marker, Cell Culture

Knockdown of NP1 reduces Kv7.2 surface expression and M-current. A , B , The proportion of Kv7.2 at the neuronal surface was determined by biotinylation (30 min at 4°C) of all surface proteins in cultured cortical neurons. The ratio of the surface over the total amount of receptor was determined by densitometric analysis and expressed as a percentage of control values. Only samples of biotinylated proteins lacking immunoreactivity to actin, which indicates contamination with intracellular proteins, were used for analyses. A , Representative Western blots of Kv7.2, Na-KATPase, and actin from total (T) and biotinylated (B) proteins. B , Quantitative analysis of the effect of gain and loss of NP1 on the surface/total levels of the Kv7.2 subunit of the voltage-gated Kv7/M K + channels. Values are mean ± SEM of shRdm ( n = 11), shNP1 ( n = 11), and pWPI-NP1 ( n = 5), from at least three independent experiments; * p
Figure Legend Snippet: Knockdown of NP1 reduces Kv7.2 surface expression and M-current. A , B , The proportion of Kv7.2 at the neuronal surface was determined by biotinylation (30 min at 4°C) of all surface proteins in cultured cortical neurons. The ratio of the surface over the total amount of receptor was determined by densitometric analysis and expressed as a percentage of control values. Only samples of biotinylated proteins lacking immunoreactivity to actin, which indicates contamination with intracellular proteins, were used for analyses. A , Representative Western blots of Kv7.2, Na-KATPase, and actin from total (T) and biotinylated (B) proteins. B , Quantitative analysis of the effect of gain and loss of NP1 on the surface/total levels of the Kv7.2 subunit of the voltage-gated Kv7/M K + channels. Values are mean ± SEM of shRdm ( n = 11), shNP1 ( n = 11), and pWPI-NP1 ( n = 5), from at least three independent experiments; * p

Techniques Used: Expressing, Cell Culture, Western Blot

12) Product Images from "Neuronal Pentraxin 1 Negatively Regulates Excitatory Synapse Density and Synaptic Plasticity"

Article Title: Neuronal Pentraxin 1 Negatively Regulates Excitatory Synapse Density and Synaptic Plasticity

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.2548-14.2015

NP1 interacts and colocalizes with Kv7.2 at presynaptic terminals of excitatory synapses and axonal growth cones. A – D , Representative Western blots of immunoprecipitation eluates separated in 6% Tris-Glycine (for high molecular weight proteins) and 4–12% Bis-Tris gels (for low molecular weight proteins). TL, Total protein lysate; Syx, syntaxin. A , Both native Kv7.2 and syntaxin 1A, but not Kv7.3, coprecipitate with NP1 in total brain extracts. B , Both native Kv7.2 and NP1 coprecipitate with syntaxin in total brain extracts. C , D , Recombinant NP1 coprecipitates Kv7.2, but not syntaxin or Kv7.3, in 293T cells transfected with NP1, 5Myc-Kv7.2, 2HA-Kv7.3, and syntaxin 1A cDNAs. Kv7.2 and Kv7.3 were immunoprecipitated with antibodies against their respective Myc and HA tags. E , F , Immunofluorescence studies and confocal microscopy were performed in high-density ( E ) or low-density ( F ) isolated cortical neurons. E , Top, Confocal sections of 0.772 μm in the z -plane showing immunofluorescence of NP1, VGLUT1, Kv7.2, and negative control (omitting primary antibodies). Bottom, Colocalization (in white) of the excitatory presynaptic marker VGLUT1 (blue) with NP1 (green) and Kv7.2 (red) is shown in a single section with the corresponding orthogonal views of the stack of confocal sections. White arrows indicate sites of colocalization. F , NP1 (green) and KV7.2 (magenta) immunofluorescence and DIC images of an isolated cortical cultured neuron (1×) with its corresponding axonal growth cone highlighted in a white square box, shown in a confocal section of 0.772 μm in the z -plane at higher (5×) magnification. The negative control for primary antibodies is shown in another growth cone on the right. The image in the bottom is the merge of NP1 and Kv7.2 immunofluorescence images in the single confocal section obtained at 5× showing colocalization (white) of NP1 and Kv7.2 in the growth cone, with the corresponding orthogonal views from its respective stack of confocal sections. Images were acquired using restricted spectral emission wavelength ranges chosen to avoid crosstalk or bleed-through between the three different channels. Scale bar, 5 μm.
Figure Legend Snippet: NP1 interacts and colocalizes with Kv7.2 at presynaptic terminals of excitatory synapses and axonal growth cones. A – D , Representative Western blots of immunoprecipitation eluates separated in 6% Tris-Glycine (for high molecular weight proteins) and 4–12% Bis-Tris gels (for low molecular weight proteins). TL, Total protein lysate; Syx, syntaxin. A , Both native Kv7.2 and syntaxin 1A, but not Kv7.3, coprecipitate with NP1 in total brain extracts. B , Both native Kv7.2 and NP1 coprecipitate with syntaxin in total brain extracts. C , D , Recombinant NP1 coprecipitates Kv7.2, but not syntaxin or Kv7.3, in 293T cells transfected with NP1, 5Myc-Kv7.2, 2HA-Kv7.3, and syntaxin 1A cDNAs. Kv7.2 and Kv7.3 were immunoprecipitated with antibodies against their respective Myc and HA tags. E , F , Immunofluorescence studies and confocal microscopy were performed in high-density ( E ) or low-density ( F ) isolated cortical neurons. E , Top, Confocal sections of 0.772 μm in the z -plane showing immunofluorescence of NP1, VGLUT1, Kv7.2, and negative control (omitting primary antibodies). Bottom, Colocalization (in white) of the excitatory presynaptic marker VGLUT1 (blue) with NP1 (green) and Kv7.2 (red) is shown in a single section with the corresponding orthogonal views of the stack of confocal sections. White arrows indicate sites of colocalization. F , NP1 (green) and KV7.2 (magenta) immunofluorescence and DIC images of an isolated cortical cultured neuron (1×) with its corresponding axonal growth cone highlighted in a white square box, shown in a confocal section of 0.772 μm in the z -plane at higher (5×) magnification. The negative control for primary antibodies is shown in another growth cone on the right. The image in the bottom is the merge of NP1 and Kv7.2 immunofluorescence images in the single confocal section obtained at 5× showing colocalization (white) of NP1 and Kv7.2 in the growth cone, with the corresponding orthogonal views from its respective stack of confocal sections. Images were acquired using restricted spectral emission wavelength ranges chosen to avoid crosstalk or bleed-through between the three different channels. Scale bar, 5 μm.

Techniques Used: Western Blot, Immunoprecipitation, Molecular Weight, Recombinant, Transfection, Immunofluorescence, Confocal Microscopy, Isolation, Negative Control, Marker, Cell Culture

Knockdown of NP1 reduces Kv7.2 surface expression and M-current. A , B , The proportion of Kv7.2 at the neuronal surface was determined by biotinylation (30 min at 4°C) of all surface proteins in cultured cortical neurons. The ratio of the surface over the total amount of receptor was determined by densitometric analysis and expressed as a percentage of control values. Only samples of biotinylated proteins lacking immunoreactivity to actin, which indicates contamination with intracellular proteins, were used for analyses. A , Representative Western blots of Kv7.2, Na-KATPase, and actin from total (T) and biotinylated (B) proteins. B , Quantitative analysis of the effect of gain and loss of NP1 on the surface/total levels of the Kv7.2 subunit of the voltage-gated Kv7/M K + channels. Values are mean ± SEM of shRdm ( n = 11), shNP1 ( n = 11), and pWPI-NP1 ( n = 5), from at least three independent experiments; * p
Figure Legend Snippet: Knockdown of NP1 reduces Kv7.2 surface expression and M-current. A , B , The proportion of Kv7.2 at the neuronal surface was determined by biotinylation (30 min at 4°C) of all surface proteins in cultured cortical neurons. The ratio of the surface over the total amount of receptor was determined by densitometric analysis and expressed as a percentage of control values. Only samples of biotinylated proteins lacking immunoreactivity to actin, which indicates contamination with intracellular proteins, were used for analyses. A , Representative Western blots of Kv7.2, Na-KATPase, and actin from total (T) and biotinylated (B) proteins. B , Quantitative analysis of the effect of gain and loss of NP1 on the surface/total levels of the Kv7.2 subunit of the voltage-gated Kv7/M K + channels. Values are mean ± SEM of shRdm ( n = 11), shNP1 ( n = 11), and pWPI-NP1 ( n = 5), from at least three independent experiments; * p

Techniques Used: Expressing, Cell Culture, Western Blot

13) Product Images from "Neuronal Pentraxin 1 Negatively Regulates Excitatory Synapse Density and Synaptic Plasticity"

Article Title: Neuronal Pentraxin 1 Negatively Regulates Excitatory Synapse Density and Synaptic Plasticity

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.2548-14.2015

NP1 interacts and colocalizes with Kv7.2 at presynaptic terminals of excitatory synapses and axonal growth cones. A – D , Representative Western blots of immunoprecipitation eluates separated in 6% Tris-Glycine (for high molecular weight proteins) and 4–12% Bis-Tris gels (for low molecular weight proteins). TL, Total protein lysate; Syx, syntaxin. A , Both native Kv7.2 and syntaxin 1A, but not Kv7.3, coprecipitate with NP1 in total brain extracts. B , Both native Kv7.2 and NP1 coprecipitate with syntaxin in total brain extracts. C , D , Recombinant NP1 coprecipitates Kv7.2, but not syntaxin or Kv7.3, in 293T cells transfected with NP1, 5Myc-Kv7.2, 2HA-Kv7.3, and syntaxin 1A cDNAs. Kv7.2 and Kv7.3 were immunoprecipitated with antibodies against their respective Myc and HA tags. E , F , Immunofluorescence studies and confocal microscopy were performed in high-density ( E ) or low-density ( F ) isolated cortical neurons. E , Top, Confocal sections of 0.772 μm in the z -plane showing immunofluorescence of NP1, VGLUT1, Kv7.2, and negative control (omitting primary antibodies). Bottom, Colocalization (in white) of the excitatory presynaptic marker VGLUT1 (blue) with NP1 (green) and Kv7.2 (red) is shown in a single section with the corresponding orthogonal views of the stack of confocal sections. White arrows indicate sites of colocalization. F , NP1 (green) and KV7.2 (magenta) immunofluorescence and DIC images of an isolated cortical cultured neuron (1×) with its corresponding axonal growth cone highlighted in a white square box, shown in a confocal section of 0.772 μm in the z -plane at higher (5×) magnification. The negative control for primary antibodies is shown in another growth cone on the right. The image in the bottom is the merge of NP1 and Kv7.2 immunofluorescence images in the single confocal section obtained at 5× showing colocalization (white) of NP1 and Kv7.2 in the growth cone, with the corresponding orthogonal views from its respective stack of confocal sections. Images were acquired using restricted spectral emission wavelength ranges chosen to avoid crosstalk or bleed-through between the three different channels. Scale bar, 5 μm.
Figure Legend Snippet: NP1 interacts and colocalizes with Kv7.2 at presynaptic terminals of excitatory synapses and axonal growth cones. A – D , Representative Western blots of immunoprecipitation eluates separated in 6% Tris-Glycine (for high molecular weight proteins) and 4–12% Bis-Tris gels (for low molecular weight proteins). TL, Total protein lysate; Syx, syntaxin. A , Both native Kv7.2 and syntaxin 1A, but not Kv7.3, coprecipitate with NP1 in total brain extracts. B , Both native Kv7.2 and NP1 coprecipitate with syntaxin in total brain extracts. C , D , Recombinant NP1 coprecipitates Kv7.2, but not syntaxin or Kv7.3, in 293T cells transfected with NP1, 5Myc-Kv7.2, 2HA-Kv7.3, and syntaxin 1A cDNAs. Kv7.2 and Kv7.3 were immunoprecipitated with antibodies against their respective Myc and HA tags. E , F , Immunofluorescence studies and confocal microscopy were performed in high-density ( E ) or low-density ( F ) isolated cortical neurons. E , Top, Confocal sections of 0.772 μm in the z -plane showing immunofluorescence of NP1, VGLUT1, Kv7.2, and negative control (omitting primary antibodies). Bottom, Colocalization (in white) of the excitatory presynaptic marker VGLUT1 (blue) with NP1 (green) and Kv7.2 (red) is shown in a single section with the corresponding orthogonal views of the stack of confocal sections. White arrows indicate sites of colocalization. F , NP1 (green) and KV7.2 (magenta) immunofluorescence and DIC images of an isolated cortical cultured neuron (1×) with its corresponding axonal growth cone highlighted in a white square box, shown in a confocal section of 0.772 μm in the z -plane at higher (5×) magnification. The negative control for primary antibodies is shown in another growth cone on the right. The image in the bottom is the merge of NP1 and Kv7.2 immunofluorescence images in the single confocal section obtained at 5× showing colocalization (white) of NP1 and Kv7.2 in the growth cone, with the corresponding orthogonal views from its respective stack of confocal sections. Images were acquired using restricted spectral emission wavelength ranges chosen to avoid crosstalk or bleed-through between the three different channels. Scale bar, 5 μm.

Techniques Used: Western Blot, Immunoprecipitation, Molecular Weight, Recombinant, Transfection, Immunofluorescence, Confocal Microscopy, Isolation, Negative Control, Marker, Cell Culture

Knockdown of NP1 reduces Kv7.2 surface expression and M-current. A , B , The proportion of Kv7.2 at the neuronal surface was determined by biotinylation (30 min at 4°C) of all surface proteins in cultured cortical neurons. The ratio of the surface over the total amount of receptor was determined by densitometric analysis and expressed as a percentage of control values. Only samples of biotinylated proteins lacking immunoreactivity to actin, which indicates contamination with intracellular proteins, were used for analyses. A , Representative Western blots of Kv7.2, Na-KATPase, and actin from total (T) and biotinylated (B) proteins. B , Quantitative analysis of the effect of gain and loss of NP1 on the surface/total levels of the Kv7.2 subunit of the voltage-gated Kv7/M K + channels. Values are mean ± SEM of shRdm ( n = 11), shNP1 ( n = 11), and pWPI-NP1 ( n = 5), from at least three independent experiments; * p
Figure Legend Snippet: Knockdown of NP1 reduces Kv7.2 surface expression and M-current. A , B , The proportion of Kv7.2 at the neuronal surface was determined by biotinylation (30 min at 4°C) of all surface proteins in cultured cortical neurons. The ratio of the surface over the total amount of receptor was determined by densitometric analysis and expressed as a percentage of control values. Only samples of biotinylated proteins lacking immunoreactivity to actin, which indicates contamination with intracellular proteins, were used for analyses. A , Representative Western blots of Kv7.2, Na-KATPase, and actin from total (T) and biotinylated (B) proteins. B , Quantitative analysis of the effect of gain and loss of NP1 on the surface/total levels of the Kv7.2 subunit of the voltage-gated Kv7/M K + channels. Values are mean ± SEM of shRdm ( n = 11), shNP1 ( n = 11), and pWPI-NP1 ( n = 5), from at least three independent experiments; * p

Techniques Used: Expressing, Cell Culture, Western Blot

14) Product Images from "Neuronal Pentraxin 1 Negatively Regulates Excitatory Synapse Density and Synaptic Plasticity"

Article Title: Neuronal Pentraxin 1 Negatively Regulates Excitatory Synapse Density and Synaptic Plasticity

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.2548-14.2015

NP1 interacts and colocalizes with Kv7.2 at presynaptic terminals of excitatory synapses and axonal growth cones. A – D , Representative Western blots of immunoprecipitation eluates separated in 6% Tris-Glycine (for high molecular weight proteins) and 4–12% Bis-Tris gels (for low molecular weight proteins). TL, Total protein lysate; Syx, syntaxin. A , Both native Kv7.2 and syntaxin 1A, but not Kv7.3, coprecipitate with NP1 in total brain extracts. B , Both native Kv7.2 and NP1 coprecipitate with syntaxin in total brain extracts. C , D , Recombinant NP1 coprecipitates Kv7.2, but not syntaxin or Kv7.3, in 293T cells transfected with NP1, 5Myc-Kv7.2, 2HA-Kv7.3, and syntaxin 1A cDNAs. Kv7.2 and Kv7.3 were immunoprecipitated with antibodies against their respective Myc and HA tags. E , F , Immunofluorescence studies and confocal microscopy were performed in high-density ( E ) or low-density ( F ) isolated cortical neurons. E , Top, Confocal sections of 0.772 μm in the z -plane showing immunofluorescence of NP1, VGLUT1, Kv7.2, and negative control (omitting primary antibodies). Bottom, Colocalization (in white) of the excitatory presynaptic marker VGLUT1 (blue) with NP1 (green) and Kv7.2 (red) is shown in a single section with the corresponding orthogonal views of the stack of confocal sections. White arrows indicate sites of colocalization. F , NP1 (green) and KV7.2 (magenta) immunofluorescence and DIC images of an isolated cortical cultured neuron (1×) with its corresponding axonal growth cone highlighted in a white square box, shown in a confocal section of 0.772 μm in the z -plane at higher (5×) magnification. The negative control for primary antibodies is shown in another growth cone on the right. The image in the bottom is the merge of NP1 and Kv7.2 immunofluorescence images in the single confocal section obtained at 5× showing colocalization (white) of NP1 and Kv7.2 in the growth cone, with the corresponding orthogonal views from its respective stack of confocal sections. Images were acquired using restricted spectral emission wavelength ranges chosen to avoid crosstalk or bleed-through between the three different channels. Scale bar, 5 μm.
Figure Legend Snippet: NP1 interacts and colocalizes with Kv7.2 at presynaptic terminals of excitatory synapses and axonal growth cones. A – D , Representative Western blots of immunoprecipitation eluates separated in 6% Tris-Glycine (for high molecular weight proteins) and 4–12% Bis-Tris gels (for low molecular weight proteins). TL, Total protein lysate; Syx, syntaxin. A , Both native Kv7.2 and syntaxin 1A, but not Kv7.3, coprecipitate with NP1 in total brain extracts. B , Both native Kv7.2 and NP1 coprecipitate with syntaxin in total brain extracts. C , D , Recombinant NP1 coprecipitates Kv7.2, but not syntaxin or Kv7.3, in 293T cells transfected with NP1, 5Myc-Kv7.2, 2HA-Kv7.3, and syntaxin 1A cDNAs. Kv7.2 and Kv7.3 were immunoprecipitated with antibodies against their respective Myc and HA tags. E , F , Immunofluorescence studies and confocal microscopy were performed in high-density ( E ) or low-density ( F ) isolated cortical neurons. E , Top, Confocal sections of 0.772 μm in the z -plane showing immunofluorescence of NP1, VGLUT1, Kv7.2, and negative control (omitting primary antibodies). Bottom, Colocalization (in white) of the excitatory presynaptic marker VGLUT1 (blue) with NP1 (green) and Kv7.2 (red) is shown in a single section with the corresponding orthogonal views of the stack of confocal sections. White arrows indicate sites of colocalization. F , NP1 (green) and KV7.2 (magenta) immunofluorescence and DIC images of an isolated cortical cultured neuron (1×) with its corresponding axonal growth cone highlighted in a white square box, shown in a confocal section of 0.772 μm in the z -plane at higher (5×) magnification. The negative control for primary antibodies is shown in another growth cone on the right. The image in the bottom is the merge of NP1 and Kv7.2 immunofluorescence images in the single confocal section obtained at 5× showing colocalization (white) of NP1 and Kv7.2 in the growth cone, with the corresponding orthogonal views from its respective stack of confocal sections. Images were acquired using restricted spectral emission wavelength ranges chosen to avoid crosstalk or bleed-through between the three different channels. Scale bar, 5 μm.

Techniques Used: Western Blot, Immunoprecipitation, Molecular Weight, Recombinant, Transfection, Immunofluorescence, Confocal Microscopy, Isolation, Negative Control, Marker, Cell Culture

Knockdown of NP1 reduces Kv7.2 surface expression and M-current. A , B , The proportion of Kv7.2 at the neuronal surface was determined by biotinylation (30 min at 4°C) of all surface proteins in cultured cortical neurons. The ratio of the surface over the total amount of receptor was determined by densitometric analysis and expressed as a percentage of control values. Only samples of biotinylated proteins lacking immunoreactivity to actin, which indicates contamination with intracellular proteins, were used for analyses. A , Representative Western blots of Kv7.2, Na-KATPase, and actin from total (T) and biotinylated (B) proteins. B , Quantitative analysis of the effect of gain and loss of NP1 on the surface/total levels of the Kv7.2 subunit of the voltage-gated Kv7/M K + channels. Values are mean ± SEM of shRdm ( n = 11), shNP1 ( n = 11), and pWPI-NP1 ( n = 5), from at least three independent experiments; * p
Figure Legend Snippet: Knockdown of NP1 reduces Kv7.2 surface expression and M-current. A , B , The proportion of Kv7.2 at the neuronal surface was determined by biotinylation (30 min at 4°C) of all surface proteins in cultured cortical neurons. The ratio of the surface over the total amount of receptor was determined by densitometric analysis and expressed as a percentage of control values. Only samples of biotinylated proteins lacking immunoreactivity to actin, which indicates contamination with intracellular proteins, were used for analyses. A , Representative Western blots of Kv7.2, Na-KATPase, and actin from total (T) and biotinylated (B) proteins. B , Quantitative analysis of the effect of gain and loss of NP1 on the surface/total levels of the Kv7.2 subunit of the voltage-gated Kv7/M K + channels. Values are mean ± SEM of shRdm ( n = 11), shNP1 ( n = 11), and pWPI-NP1 ( n = 5), from at least three independent experiments; * p

Techniques Used: Expressing, Cell Culture, Western Blot

15) Product Images from "Neuronal Pentraxin 1 Negatively Regulates Excitatory Synapse Density and Synaptic Plasticity"

Article Title: Neuronal Pentraxin 1 Negatively Regulates Excitatory Synapse Density and Synaptic Plasticity

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.2548-14.2015

NP1 interacts and colocalizes with Kv7.2 at presynaptic terminals of excitatory synapses and axonal growth cones. A – D , Representative Western blots of immunoprecipitation eluates separated in 6% Tris-Glycine (for high molecular weight proteins) and 4–12% Bis-Tris gels (for low molecular weight proteins). TL, Total protein lysate; Syx, syntaxin. A , Both native Kv7.2 and syntaxin 1A, but not Kv7.3, coprecipitate with NP1 in total brain extracts. B , Both native Kv7.2 and NP1 coprecipitate with syntaxin in total brain extracts. C , D , Recombinant NP1 coprecipitates Kv7.2, but not syntaxin or Kv7.3, in 293T cells transfected with NP1, 5Myc-Kv7.2, 2HA-Kv7.3, and syntaxin 1A cDNAs. Kv7.2 and Kv7.3 were immunoprecipitated with antibodies against their respective Myc and HA tags. E , F , Immunofluorescence studies and confocal microscopy were performed in high-density ( E ) or low-density ( F ) isolated cortical neurons. E , Top, Confocal sections of 0.772 μm in the z -plane showing immunofluorescence of NP1, VGLUT1, Kv7.2, and negative control (omitting primary antibodies). Bottom, Colocalization (in white) of the excitatory presynaptic marker VGLUT1 (blue) with NP1 (green) and Kv7.2 (red) is shown in a single section with the corresponding orthogonal views of the stack of confocal sections. White arrows indicate sites of colocalization. F , NP1 (green) and KV7.2 (magenta) immunofluorescence and DIC images of an isolated cortical cultured neuron (1×) with its corresponding axonal growth cone highlighted in a white square box, shown in a confocal section of 0.772 μm in the z -plane at higher (5×) magnification. The negative control for primary antibodies is shown in another growth cone on the right. The image in the bottom is the merge of NP1 and Kv7.2 immunofluorescence images in the single confocal section obtained at 5× showing colocalization (white) of NP1 and Kv7.2 in the growth cone, with the corresponding orthogonal views from its respective stack of confocal sections. Images were acquired using restricted spectral emission wavelength ranges chosen to avoid crosstalk or bleed-through between the three different channels. Scale bar, 5 μm.
Figure Legend Snippet: NP1 interacts and colocalizes with Kv7.2 at presynaptic terminals of excitatory synapses and axonal growth cones. A – D , Representative Western blots of immunoprecipitation eluates separated in 6% Tris-Glycine (for high molecular weight proteins) and 4–12% Bis-Tris gels (for low molecular weight proteins). TL, Total protein lysate; Syx, syntaxin. A , Both native Kv7.2 and syntaxin 1A, but not Kv7.3, coprecipitate with NP1 in total brain extracts. B , Both native Kv7.2 and NP1 coprecipitate with syntaxin in total brain extracts. C , D , Recombinant NP1 coprecipitates Kv7.2, but not syntaxin or Kv7.3, in 293T cells transfected with NP1, 5Myc-Kv7.2, 2HA-Kv7.3, and syntaxin 1A cDNAs. Kv7.2 and Kv7.3 were immunoprecipitated with antibodies against their respective Myc and HA tags. E , F , Immunofluorescence studies and confocal microscopy were performed in high-density ( E ) or low-density ( F ) isolated cortical neurons. E , Top, Confocal sections of 0.772 μm in the z -plane showing immunofluorescence of NP1, VGLUT1, Kv7.2, and negative control (omitting primary antibodies). Bottom, Colocalization (in white) of the excitatory presynaptic marker VGLUT1 (blue) with NP1 (green) and Kv7.2 (red) is shown in a single section with the corresponding orthogonal views of the stack of confocal sections. White arrows indicate sites of colocalization. F , NP1 (green) and KV7.2 (magenta) immunofluorescence and DIC images of an isolated cortical cultured neuron (1×) with its corresponding axonal growth cone highlighted in a white square box, shown in a confocal section of 0.772 μm in the z -plane at higher (5×) magnification. The negative control for primary antibodies is shown in another growth cone on the right. The image in the bottom is the merge of NP1 and Kv7.2 immunofluorescence images in the single confocal section obtained at 5× showing colocalization (white) of NP1 and Kv7.2 in the growth cone, with the corresponding orthogonal views from its respective stack of confocal sections. Images were acquired using restricted spectral emission wavelength ranges chosen to avoid crosstalk or bleed-through between the three different channels. Scale bar, 5 μm.

Techniques Used: Western Blot, Immunoprecipitation, Molecular Weight, Recombinant, Transfection, Immunofluorescence, Confocal Microscopy, Isolation, Negative Control, Marker, Cell Culture

Knockdown of NP1 reduces Kv7.2 surface expression and M-current. A , B , The proportion of Kv7.2 at the neuronal surface was determined by biotinylation (30 min at 4°C) of all surface proteins in cultured cortical neurons. The ratio of the surface over the total amount of receptor was determined by densitometric analysis and expressed as a percentage of control values. Only samples of biotinylated proteins lacking immunoreactivity to actin, which indicates contamination with intracellular proteins, were used for analyses. A , Representative Western blots of Kv7.2, Na-KATPase, and actin from total (T) and biotinylated (B) proteins. B , Quantitative analysis of the effect of gain and loss of NP1 on the surface/total levels of the Kv7.2 subunit of the voltage-gated Kv7/M K + channels. Values are mean ± SEM of shRdm ( n = 11), shNP1 ( n = 11), and pWPI-NP1 ( n = 5), from at least three independent experiments; * p
Figure Legend Snippet: Knockdown of NP1 reduces Kv7.2 surface expression and M-current. A , B , The proportion of Kv7.2 at the neuronal surface was determined by biotinylation (30 min at 4°C) of all surface proteins in cultured cortical neurons. The ratio of the surface over the total amount of receptor was determined by densitometric analysis and expressed as a percentage of control values. Only samples of biotinylated proteins lacking immunoreactivity to actin, which indicates contamination with intracellular proteins, were used for analyses. A , Representative Western blots of Kv7.2, Na-KATPase, and actin from total (T) and biotinylated (B) proteins. B , Quantitative analysis of the effect of gain and loss of NP1 on the surface/total levels of the Kv7.2 subunit of the voltage-gated Kv7/M K + channels. Values are mean ± SEM of shRdm ( n = 11), shNP1 ( n = 11), and pWPI-NP1 ( n = 5), from at least three independent experiments; * p

Techniques Used: Expressing, Cell Culture, Western Blot

16) Product Images from "Neuronal Pentraxin 1 Negatively Regulates Excitatory Synapse Density and Synaptic Plasticity"

Article Title: Neuronal Pentraxin 1 Negatively Regulates Excitatory Synapse Density and Synaptic Plasticity

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.2548-14.2015

NP1 interacts and colocalizes with Kv7.2 at presynaptic terminals of excitatory synapses and axonal growth cones. A – D , Representative Western blots of immunoprecipitation eluates separated in 6% Tris-Glycine (for high molecular weight proteins) and 4–12% Bis-Tris gels (for low molecular weight proteins). TL, Total protein lysate; Syx, syntaxin. A , Both native Kv7.2 and syntaxin 1A, but not Kv7.3, coprecipitate with NP1 in total brain extracts. B , Both native Kv7.2 and NP1 coprecipitate with syntaxin in total brain extracts. C , D , Recombinant NP1 coprecipitates Kv7.2, but not syntaxin or Kv7.3, in 293T cells transfected with NP1, 5Myc-Kv7.2, 2HA-Kv7.3, and syntaxin 1A cDNAs. Kv7.2 and Kv7.3 were immunoprecipitated with antibodies against their respective Myc and HA tags. E , F , Immunofluorescence studies and confocal microscopy were performed in high-density ( E ) or low-density ( F ) isolated cortical neurons. E , Top, Confocal sections of 0.772 μm in the z -plane showing immunofluorescence of NP1, VGLUT1, Kv7.2, and negative control (omitting primary antibodies). Bottom, Colocalization (in white) of the excitatory presynaptic marker VGLUT1 (blue) with NP1 (green) and Kv7.2 (red) is shown in a single section with the corresponding orthogonal views of the stack of confocal sections. White arrows indicate sites of colocalization. F , NP1 (green) and KV7.2 (magenta) immunofluorescence and DIC images of an isolated cortical cultured neuron (1×) with its corresponding axonal growth cone highlighted in a white square box, shown in a confocal section of 0.772 μm in the z -plane at higher (5×) magnification. The negative control for primary antibodies is shown in another growth cone on the right. The image in the bottom is the merge of NP1 and Kv7.2 immunofluorescence images in the single confocal section obtained at 5× showing colocalization (white) of NP1 and Kv7.2 in the growth cone, with the corresponding orthogonal views from its respective stack of confocal sections. Images were acquired using restricted spectral emission wavelength ranges chosen to avoid crosstalk or bleed-through between the three different channels. Scale bar, 5 μm.
Figure Legend Snippet: NP1 interacts and colocalizes with Kv7.2 at presynaptic terminals of excitatory synapses and axonal growth cones. A – D , Representative Western blots of immunoprecipitation eluates separated in 6% Tris-Glycine (for high molecular weight proteins) and 4–12% Bis-Tris gels (for low molecular weight proteins). TL, Total protein lysate; Syx, syntaxin. A , Both native Kv7.2 and syntaxin 1A, but not Kv7.3, coprecipitate with NP1 in total brain extracts. B , Both native Kv7.2 and NP1 coprecipitate with syntaxin in total brain extracts. C , D , Recombinant NP1 coprecipitates Kv7.2, but not syntaxin or Kv7.3, in 293T cells transfected with NP1, 5Myc-Kv7.2, 2HA-Kv7.3, and syntaxin 1A cDNAs. Kv7.2 and Kv7.3 were immunoprecipitated with antibodies against their respective Myc and HA tags. E , F , Immunofluorescence studies and confocal microscopy were performed in high-density ( E ) or low-density ( F ) isolated cortical neurons. E , Top, Confocal sections of 0.772 μm in the z -plane showing immunofluorescence of NP1, VGLUT1, Kv7.2, and negative control (omitting primary antibodies). Bottom, Colocalization (in white) of the excitatory presynaptic marker VGLUT1 (blue) with NP1 (green) and Kv7.2 (red) is shown in a single section with the corresponding orthogonal views of the stack of confocal sections. White arrows indicate sites of colocalization. F , NP1 (green) and KV7.2 (magenta) immunofluorescence and DIC images of an isolated cortical cultured neuron (1×) with its corresponding axonal growth cone highlighted in a white square box, shown in a confocal section of 0.772 μm in the z -plane at higher (5×) magnification. The negative control for primary antibodies is shown in another growth cone on the right. The image in the bottom is the merge of NP1 and Kv7.2 immunofluorescence images in the single confocal section obtained at 5× showing colocalization (white) of NP1 and Kv7.2 in the growth cone, with the corresponding orthogonal views from its respective stack of confocal sections. Images were acquired using restricted spectral emission wavelength ranges chosen to avoid crosstalk or bleed-through between the three different channels. Scale bar, 5 μm.

Techniques Used: Western Blot, Immunoprecipitation, Molecular Weight, Recombinant, Transfection, Immunofluorescence, Confocal Microscopy, Isolation, Negative Control, Marker, Cell Culture

Knockdown of NP1 reduces Kv7.2 surface expression and M-current. A , B , The proportion of Kv7.2 at the neuronal surface was determined by biotinylation (30 min at 4°C) of all surface proteins in cultured cortical neurons. The ratio of the surface over the total amount of receptor was determined by densitometric analysis and expressed as a percentage of control values. Only samples of biotinylated proteins lacking immunoreactivity to actin, which indicates contamination with intracellular proteins, were used for analyses. A , Representative Western blots of Kv7.2, Na-KATPase, and actin from total (T) and biotinylated (B) proteins. B , Quantitative analysis of the effect of gain and loss of NP1 on the surface/total levels of the Kv7.2 subunit of the voltage-gated Kv7/M K + channels. Values are mean ± SEM of shRdm ( n = 11), shNP1 ( n = 11), and pWPI-NP1 ( n = 5), from at least three independent experiments; * p
Figure Legend Snippet: Knockdown of NP1 reduces Kv7.2 surface expression and M-current. A , B , The proportion of Kv7.2 at the neuronal surface was determined by biotinylation (30 min at 4°C) of all surface proteins in cultured cortical neurons. The ratio of the surface over the total amount of receptor was determined by densitometric analysis and expressed as a percentage of control values. Only samples of biotinylated proteins lacking immunoreactivity to actin, which indicates contamination with intracellular proteins, were used for analyses. A , Representative Western blots of Kv7.2, Na-KATPase, and actin from total (T) and biotinylated (B) proteins. B , Quantitative analysis of the effect of gain and loss of NP1 on the surface/total levels of the Kv7.2 subunit of the voltage-gated Kv7/M K + channels. Values are mean ± SEM of shRdm ( n = 11), shNP1 ( n = 11), and pWPI-NP1 ( n = 5), from at least three independent experiments; * p

Techniques Used: Expressing, Cell Culture, Western Blot

17) Product Images from "Neuronal Pentraxin 1 Negatively Regulates Excitatory Synapse Density and Synaptic Plasticity"

Article Title: Neuronal Pentraxin 1 Negatively Regulates Excitatory Synapse Density and Synaptic Plasticity

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.2548-14.2015

NP1 interacts and colocalizes with Kv7.2 at presynaptic terminals of excitatory synapses and axonal growth cones. A – D , Representative Western blots of immunoprecipitation eluates separated in 6% Tris-Glycine (for high molecular weight proteins) and 4–12% Bis-Tris gels (for low molecular weight proteins). TL, Total protein lysate; Syx, syntaxin. A , Both native Kv7.2 and syntaxin 1A, but not Kv7.3, coprecipitate with NP1 in total brain extracts. B , Both native Kv7.2 and NP1 coprecipitate with syntaxin in total brain extracts. C , D , Recombinant NP1 coprecipitates Kv7.2, but not syntaxin or Kv7.3, in 293T cells transfected with NP1, 5Myc-Kv7.2, 2HA-Kv7.3, and syntaxin 1A cDNAs. Kv7.2 and Kv7.3 were immunoprecipitated with antibodies against their respective Myc and HA tags. E , F , Immunofluorescence studies and confocal microscopy were performed in high-density ( E ) or low-density ( F ) isolated cortical neurons. E , Top, Confocal sections of 0.772 μm in the z -plane showing immunofluorescence of NP1, VGLUT1, Kv7.2, and negative control (omitting primary antibodies). Bottom, Colocalization (in white) of the excitatory presynaptic marker VGLUT1 (blue) with NP1 (green) and Kv7.2 (red) is shown in a single section with the corresponding orthogonal views of the stack of confocal sections. White arrows indicate sites of colocalization. F , NP1 (green) and KV7.2 (magenta) immunofluorescence and DIC images of an isolated cortical cultured neuron (1×) with its corresponding axonal growth cone highlighted in a white square box, shown in a confocal section of 0.772 μm in the z -plane at higher (5×) magnification. The negative control for primary antibodies is shown in another growth cone on the right. The image in the bottom is the merge of NP1 and Kv7.2 immunofluorescence images in the single confocal section obtained at 5× showing colocalization (white) of NP1 and Kv7.2 in the growth cone, with the corresponding orthogonal views from its respective stack of confocal sections. Images were acquired using restricted spectral emission wavelength ranges chosen to avoid crosstalk or bleed-through between the three different channels. Scale bar, 5 μm.
Figure Legend Snippet: NP1 interacts and colocalizes with Kv7.2 at presynaptic terminals of excitatory synapses and axonal growth cones. A – D , Representative Western blots of immunoprecipitation eluates separated in 6% Tris-Glycine (for high molecular weight proteins) and 4–12% Bis-Tris gels (for low molecular weight proteins). TL, Total protein lysate; Syx, syntaxin. A , Both native Kv7.2 and syntaxin 1A, but not Kv7.3, coprecipitate with NP1 in total brain extracts. B , Both native Kv7.2 and NP1 coprecipitate with syntaxin in total brain extracts. C , D , Recombinant NP1 coprecipitates Kv7.2, but not syntaxin or Kv7.3, in 293T cells transfected with NP1, 5Myc-Kv7.2, 2HA-Kv7.3, and syntaxin 1A cDNAs. Kv7.2 and Kv7.3 were immunoprecipitated with antibodies against their respective Myc and HA tags. E , F , Immunofluorescence studies and confocal microscopy were performed in high-density ( E ) or low-density ( F ) isolated cortical neurons. E , Top, Confocal sections of 0.772 μm in the z -plane showing immunofluorescence of NP1, VGLUT1, Kv7.2, and negative control (omitting primary antibodies). Bottom, Colocalization (in white) of the excitatory presynaptic marker VGLUT1 (blue) with NP1 (green) and Kv7.2 (red) is shown in a single section with the corresponding orthogonal views of the stack of confocal sections. White arrows indicate sites of colocalization. F , NP1 (green) and KV7.2 (magenta) immunofluorescence and DIC images of an isolated cortical cultured neuron (1×) with its corresponding axonal growth cone highlighted in a white square box, shown in a confocal section of 0.772 μm in the z -plane at higher (5×) magnification. The negative control for primary antibodies is shown in another growth cone on the right. The image in the bottom is the merge of NP1 and Kv7.2 immunofluorescence images in the single confocal section obtained at 5× showing colocalization (white) of NP1 and Kv7.2 in the growth cone, with the corresponding orthogonal views from its respective stack of confocal sections. Images were acquired using restricted spectral emission wavelength ranges chosen to avoid crosstalk or bleed-through between the three different channels. Scale bar, 5 μm.

Techniques Used: Western Blot, Immunoprecipitation, Molecular Weight, Recombinant, Transfection, Immunofluorescence, Confocal Microscopy, Isolation, Negative Control, Marker, Cell Culture

Knockdown of NP1 reduces Kv7.2 surface expression and M-current. A , B , The proportion of Kv7.2 at the neuronal surface was determined by biotinylation (30 min at 4°C) of all surface proteins in cultured cortical neurons. The ratio of the surface over the total amount of receptor was determined by densitometric analysis and expressed as a percentage of control values. Only samples of biotinylated proteins lacking immunoreactivity to actin, which indicates contamination with intracellular proteins, were used for analyses. A , Representative Western blots of Kv7.2, Na-KATPase, and actin from total (T) and biotinylated (B) proteins. B , Quantitative analysis of the effect of gain and loss of NP1 on the surface/total levels of the Kv7.2 subunit of the voltage-gated Kv7/M K + channels. Values are mean ± SEM of shRdm ( n = 11), shNP1 ( n = 11), and pWPI-NP1 ( n = 5), from at least three independent experiments; * p
Figure Legend Snippet: Knockdown of NP1 reduces Kv7.2 surface expression and M-current. A , B , The proportion of Kv7.2 at the neuronal surface was determined by biotinylation (30 min at 4°C) of all surface proteins in cultured cortical neurons. The ratio of the surface over the total amount of receptor was determined by densitometric analysis and expressed as a percentage of control values. Only samples of biotinylated proteins lacking immunoreactivity to actin, which indicates contamination with intracellular proteins, were used for analyses. A , Representative Western blots of Kv7.2, Na-KATPase, and actin from total (T) and biotinylated (B) proteins. B , Quantitative analysis of the effect of gain and loss of NP1 on the surface/total levels of the Kv7.2 subunit of the voltage-gated Kv7/M K + channels. Values are mean ± SEM of shRdm ( n = 11), shNP1 ( n = 11), and pWPI-NP1 ( n = 5), from at least three independent experiments; * p

Techniques Used: Expressing, Cell Culture, Western Blot

18) Product Images from "Neuronal Pentraxin 1 Negatively Regulates Excitatory Synapse Density and Synaptic Plasticity"

Article Title: Neuronal Pentraxin 1 Negatively Regulates Excitatory Synapse Density and Synaptic Plasticity

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.2548-14.2015

NP1 interacts and colocalizes with Kv7.2 at presynaptic terminals of excitatory synapses and axonal growth cones. A – D , Representative Western blots of immunoprecipitation eluates separated in 6% Tris-Glycine (for high molecular weight proteins) and 4–12% Bis-Tris gels (for low molecular weight proteins). TL, Total protein lysate; Syx, syntaxin. A , Both native Kv7.2 and syntaxin 1A, but not Kv7.3, coprecipitate with NP1 in total brain extracts. B , Both native Kv7.2 and NP1 coprecipitate with syntaxin in total brain extracts. C , D , Recombinant NP1 coprecipitates Kv7.2, but not syntaxin or Kv7.3, in 293T cells transfected with NP1, 5Myc-Kv7.2, 2HA-Kv7.3, and syntaxin 1A cDNAs. Kv7.2 and Kv7.3 were immunoprecipitated with antibodies against their respective Myc and HA tags. E , F , Immunofluorescence studies and confocal microscopy were performed in high-density ( E ) or low-density ( F ) isolated cortical neurons. E , Top, Confocal sections of 0.772 μm in the z -plane showing immunofluorescence of NP1, VGLUT1, Kv7.2, and negative control (omitting primary antibodies). Bottom, Colocalization (in white) of the excitatory presynaptic marker VGLUT1 (blue) with NP1 (green) and Kv7.2 (red) is shown in a single section with the corresponding orthogonal views of the stack of confocal sections. White arrows indicate sites of colocalization. F , NP1 (green) and KV7.2 (magenta) immunofluorescence and DIC images of an isolated cortical cultured neuron (1×) with its corresponding axonal growth cone highlighted in a white square box, shown in a confocal section of 0.772 μm in the z -plane at higher (5×) magnification. The negative control for primary antibodies is shown in another growth cone on the right. The image in the bottom is the merge of NP1 and Kv7.2 immunofluorescence images in the single confocal section obtained at 5× showing colocalization (white) of NP1 and Kv7.2 in the growth cone, with the corresponding orthogonal views from its respective stack of confocal sections. Images were acquired using restricted spectral emission wavelength ranges chosen to avoid crosstalk or bleed-through between the three different channels. Scale bar, 5 μm.
Figure Legend Snippet: NP1 interacts and colocalizes with Kv7.2 at presynaptic terminals of excitatory synapses and axonal growth cones. A – D , Representative Western blots of immunoprecipitation eluates separated in 6% Tris-Glycine (for high molecular weight proteins) and 4–12% Bis-Tris gels (for low molecular weight proteins). TL, Total protein lysate; Syx, syntaxin. A , Both native Kv7.2 and syntaxin 1A, but not Kv7.3, coprecipitate with NP1 in total brain extracts. B , Both native Kv7.2 and NP1 coprecipitate with syntaxin in total brain extracts. C , D , Recombinant NP1 coprecipitates Kv7.2, but not syntaxin or Kv7.3, in 293T cells transfected with NP1, 5Myc-Kv7.2, 2HA-Kv7.3, and syntaxin 1A cDNAs. Kv7.2 and Kv7.3 were immunoprecipitated with antibodies against their respective Myc and HA tags. E , F , Immunofluorescence studies and confocal microscopy were performed in high-density ( E ) or low-density ( F ) isolated cortical neurons. E , Top, Confocal sections of 0.772 μm in the z -plane showing immunofluorescence of NP1, VGLUT1, Kv7.2, and negative control (omitting primary antibodies). Bottom, Colocalization (in white) of the excitatory presynaptic marker VGLUT1 (blue) with NP1 (green) and Kv7.2 (red) is shown in a single section with the corresponding orthogonal views of the stack of confocal sections. White arrows indicate sites of colocalization. F , NP1 (green) and KV7.2 (magenta) immunofluorescence and DIC images of an isolated cortical cultured neuron (1×) with its corresponding axonal growth cone highlighted in a white square box, shown in a confocal section of 0.772 μm in the z -plane at higher (5×) magnification. The negative control for primary antibodies is shown in another growth cone on the right. The image in the bottom is the merge of NP1 and Kv7.2 immunofluorescence images in the single confocal section obtained at 5× showing colocalization (white) of NP1 and Kv7.2 in the growth cone, with the corresponding orthogonal views from its respective stack of confocal sections. Images were acquired using restricted spectral emission wavelength ranges chosen to avoid crosstalk or bleed-through between the three different channels. Scale bar, 5 μm.

Techniques Used: Western Blot, Immunoprecipitation, Molecular Weight, Recombinant, Transfection, Immunofluorescence, Confocal Microscopy, Isolation, Negative Control, Marker, Cell Culture

Knockdown of NP1 reduces Kv7.2 surface expression and M-current. A , B , The proportion of Kv7.2 at the neuronal surface was determined by biotinylation (30 min at 4°C) of all surface proteins in cultured cortical neurons. The ratio of the surface over the total amount of receptor was determined by densitometric analysis and expressed as a percentage of control values. Only samples of biotinylated proteins lacking immunoreactivity to actin, which indicates contamination with intracellular proteins, were used for analyses. A , Representative Western blots of Kv7.2, Na-KATPase, and actin from total (T) and biotinylated (B) proteins. B , Quantitative analysis of the effect of gain and loss of NP1 on the surface/total levels of the Kv7.2 subunit of the voltage-gated Kv7/M K + channels. Values are mean ± SEM of shRdm ( n = 11), shNP1 ( n = 11), and pWPI-NP1 ( n = 5), from at least three independent experiments; * p
Figure Legend Snippet: Knockdown of NP1 reduces Kv7.2 surface expression and M-current. A , B , The proportion of Kv7.2 at the neuronal surface was determined by biotinylation (30 min at 4°C) of all surface proteins in cultured cortical neurons. The ratio of the surface over the total amount of receptor was determined by densitometric analysis and expressed as a percentage of control values. Only samples of biotinylated proteins lacking immunoreactivity to actin, which indicates contamination with intracellular proteins, were used for analyses. A , Representative Western blots of Kv7.2, Na-KATPase, and actin from total (T) and biotinylated (B) proteins. B , Quantitative analysis of the effect of gain and loss of NP1 on the surface/total levels of the Kv7.2 subunit of the voltage-gated Kv7/M K + channels. Values are mean ± SEM of shRdm ( n = 11), shNP1 ( n = 11), and pWPI-NP1 ( n = 5), from at least three independent experiments; * p

Techniques Used: Expressing, Cell Culture, Western Blot

19) Product Images from "Neuronal Pentraxin 1 Negatively Regulates Excitatory Synapse Density and Synaptic Plasticity"

Article Title: Neuronal Pentraxin 1 Negatively Regulates Excitatory Synapse Density and Synaptic Plasticity

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.2548-14.2015

NP1 interacts and colocalizes with Kv7.2 at presynaptic terminals of excitatory synapses and axonal growth cones. A – D , Representative Western blots of immunoprecipitation eluates separated in 6% Tris-Glycine (for high molecular weight proteins) and 4–12% Bis-Tris gels (for low molecular weight proteins). TL, Total protein lysate; Syx, syntaxin. A , Both native Kv7.2 and syntaxin 1A, but not Kv7.3, coprecipitate with NP1 in total brain extracts. B , Both native Kv7.2 and NP1 coprecipitate with syntaxin in total brain extracts. C , D , Recombinant NP1 coprecipitates Kv7.2, but not syntaxin or Kv7.3, in 293T cells transfected with NP1, 5Myc-Kv7.2, 2HA-Kv7.3, and syntaxin 1A cDNAs. Kv7.2 and Kv7.3 were immunoprecipitated with antibodies against their respective Myc and HA tags. E , F , Immunofluorescence studies and confocal microscopy were performed in high-density ( E ) or low-density ( F ) isolated cortical neurons. E , Top, Confocal sections of 0.772 μm in the z -plane showing immunofluorescence of NP1, VGLUT1, Kv7.2, and negative control (omitting primary antibodies). Bottom, Colocalization (in white) of the excitatory presynaptic marker VGLUT1 (blue) with NP1 (green) and Kv7.2 (red) is shown in a single section with the corresponding orthogonal views of the stack of confocal sections. White arrows indicate sites of colocalization. F , NP1 (green) and KV7.2 (magenta) immunofluorescence and DIC images of an isolated cortical cultured neuron (1×) with its corresponding axonal growth cone highlighted in a white square box, shown in a confocal section of 0.772 μm in the z -plane at higher (5×) magnification. The negative control for primary antibodies is shown in another growth cone on the right. The image in the bottom is the merge of NP1 and Kv7.2 immunofluorescence images in the single confocal section obtained at 5× showing colocalization (white) of NP1 and Kv7.2 in the growth cone, with the corresponding orthogonal views from its respective stack of confocal sections. Images were acquired using restricted spectral emission wavelength ranges chosen to avoid crosstalk or bleed-through between the three different channels. Scale bar, 5 μm.
Figure Legend Snippet: NP1 interacts and colocalizes with Kv7.2 at presynaptic terminals of excitatory synapses and axonal growth cones. A – D , Representative Western blots of immunoprecipitation eluates separated in 6% Tris-Glycine (for high molecular weight proteins) and 4–12% Bis-Tris gels (for low molecular weight proteins). TL, Total protein lysate; Syx, syntaxin. A , Both native Kv7.2 and syntaxin 1A, but not Kv7.3, coprecipitate with NP1 in total brain extracts. B , Both native Kv7.2 and NP1 coprecipitate with syntaxin in total brain extracts. C , D , Recombinant NP1 coprecipitates Kv7.2, but not syntaxin or Kv7.3, in 293T cells transfected with NP1, 5Myc-Kv7.2, 2HA-Kv7.3, and syntaxin 1A cDNAs. Kv7.2 and Kv7.3 were immunoprecipitated with antibodies against their respective Myc and HA tags. E , F , Immunofluorescence studies and confocal microscopy were performed in high-density ( E ) or low-density ( F ) isolated cortical neurons. E , Top, Confocal sections of 0.772 μm in the z -plane showing immunofluorescence of NP1, VGLUT1, Kv7.2, and negative control (omitting primary antibodies). Bottom, Colocalization (in white) of the excitatory presynaptic marker VGLUT1 (blue) with NP1 (green) and Kv7.2 (red) is shown in a single section with the corresponding orthogonal views of the stack of confocal sections. White arrows indicate sites of colocalization. F , NP1 (green) and KV7.2 (magenta) immunofluorescence and DIC images of an isolated cortical cultured neuron (1×) with its corresponding axonal growth cone highlighted in a white square box, shown in a confocal section of 0.772 μm in the z -plane at higher (5×) magnification. The negative control for primary antibodies is shown in another growth cone on the right. The image in the bottom is the merge of NP1 and Kv7.2 immunofluorescence images in the single confocal section obtained at 5× showing colocalization (white) of NP1 and Kv7.2 in the growth cone, with the corresponding orthogonal views from its respective stack of confocal sections. Images were acquired using restricted spectral emission wavelength ranges chosen to avoid crosstalk or bleed-through between the three different channels. Scale bar, 5 μm.

Techniques Used: Western Blot, Immunoprecipitation, Molecular Weight, Recombinant, Transfection, Immunofluorescence, Confocal Microscopy, Isolation, Negative Control, Marker, Cell Culture

Knockdown of NP1 reduces Kv7.2 surface expression and M-current. A , B , The proportion of Kv7.2 at the neuronal surface was determined by biotinylation (30 min at 4°C) of all surface proteins in cultured cortical neurons. The ratio of the surface over the total amount of receptor was determined by densitometric analysis and expressed as a percentage of control values. Only samples of biotinylated proteins lacking immunoreactivity to actin, which indicates contamination with intracellular proteins, were used for analyses. A , Representative Western blots of Kv7.2, Na-KATPase, and actin from total (T) and biotinylated (B) proteins. B , Quantitative analysis of the effect of gain and loss of NP1 on the surface/total levels of the Kv7.2 subunit of the voltage-gated Kv7/M K + channels. Values are mean ± SEM of shRdm ( n = 11), shNP1 ( n = 11), and pWPI-NP1 ( n = 5), from at least three independent experiments; * p
Figure Legend Snippet: Knockdown of NP1 reduces Kv7.2 surface expression and M-current. A , B , The proportion of Kv7.2 at the neuronal surface was determined by biotinylation (30 min at 4°C) of all surface proteins in cultured cortical neurons. The ratio of the surface over the total amount of receptor was determined by densitometric analysis and expressed as a percentage of control values. Only samples of biotinylated proteins lacking immunoreactivity to actin, which indicates contamination with intracellular proteins, were used for analyses. A , Representative Western blots of Kv7.2, Na-KATPase, and actin from total (T) and biotinylated (B) proteins. B , Quantitative analysis of the effect of gain and loss of NP1 on the surface/total levels of the Kv7.2 subunit of the voltage-gated Kv7/M K + channels. Values are mean ± SEM of shRdm ( n = 11), shNP1 ( n = 11), and pWPI-NP1 ( n = 5), from at least three independent experiments; * p

Techniques Used: Expressing, Cell Culture, Western Blot

20) Product Images from "Neuronal Pentraxin 1 Negatively Regulates Excitatory Synapse Density and Synaptic Plasticity"

Article Title: Neuronal Pentraxin 1 Negatively Regulates Excitatory Synapse Density and Synaptic Plasticity

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.2548-14.2015

NP1 interacts and colocalizes with Kv7.2 at presynaptic terminals of excitatory synapses and axonal growth cones. A – D , Representative Western blots of immunoprecipitation eluates separated in 6% Tris-Glycine (for high molecular weight proteins) and 4–12% Bis-Tris gels (for low molecular weight proteins). TL, Total protein lysate; Syx, syntaxin. A , Both native Kv7.2 and syntaxin 1A, but not Kv7.3, coprecipitate with NP1 in total brain extracts. B , Both native Kv7.2 and NP1 coprecipitate with syntaxin in total brain extracts. C , D , Recombinant NP1 coprecipitates Kv7.2, but not syntaxin or Kv7.3, in 293T cells transfected with NP1, 5Myc-Kv7.2, 2HA-Kv7.3, and syntaxin 1A cDNAs. Kv7.2 and Kv7.3 were immunoprecipitated with antibodies against their respective Myc and HA tags. E , F , Immunofluorescence studies and confocal microscopy were performed in high-density ( E ) or low-density ( F ) isolated cortical neurons. E , Top, Confocal sections of 0.772 μm in the z -plane showing immunofluorescence of NP1, VGLUT1, Kv7.2, and negative control (omitting primary antibodies). Bottom, Colocalization (in white) of the excitatory presynaptic marker VGLUT1 (blue) with NP1 (green) and Kv7.2 (red) is shown in a single section with the corresponding orthogonal views of the stack of confocal sections. White arrows indicate sites of colocalization. F , NP1 (green) and KV7.2 (magenta) immunofluorescence and DIC images of an isolated cortical cultured neuron (1×) with its corresponding axonal growth cone highlighted in a white square box, shown in a confocal section of 0.772 μm in the z -plane at higher (5×) magnification. The negative control for primary antibodies is shown in another growth cone on the right. The image in the bottom is the merge of NP1 and Kv7.2 immunofluorescence images in the single confocal section obtained at 5× showing colocalization (white) of NP1 and Kv7.2 in the growth cone, with the corresponding orthogonal views from its respective stack of confocal sections. Images were acquired using restricted spectral emission wavelength ranges chosen to avoid crosstalk or bleed-through between the three different channels. Scale bar, 5 μm.
Figure Legend Snippet: NP1 interacts and colocalizes with Kv7.2 at presynaptic terminals of excitatory synapses and axonal growth cones. A – D , Representative Western blots of immunoprecipitation eluates separated in 6% Tris-Glycine (for high molecular weight proteins) and 4–12% Bis-Tris gels (for low molecular weight proteins). TL, Total protein lysate; Syx, syntaxin. A , Both native Kv7.2 and syntaxin 1A, but not Kv7.3, coprecipitate with NP1 in total brain extracts. B , Both native Kv7.2 and NP1 coprecipitate with syntaxin in total brain extracts. C , D , Recombinant NP1 coprecipitates Kv7.2, but not syntaxin or Kv7.3, in 293T cells transfected with NP1, 5Myc-Kv7.2, 2HA-Kv7.3, and syntaxin 1A cDNAs. Kv7.2 and Kv7.3 were immunoprecipitated with antibodies against their respective Myc and HA tags. E , F , Immunofluorescence studies and confocal microscopy were performed in high-density ( E ) or low-density ( F ) isolated cortical neurons. E , Top, Confocal sections of 0.772 μm in the z -plane showing immunofluorescence of NP1, VGLUT1, Kv7.2, and negative control (omitting primary antibodies). Bottom, Colocalization (in white) of the excitatory presynaptic marker VGLUT1 (blue) with NP1 (green) and Kv7.2 (red) is shown in a single section with the corresponding orthogonal views of the stack of confocal sections. White arrows indicate sites of colocalization. F , NP1 (green) and KV7.2 (magenta) immunofluorescence and DIC images of an isolated cortical cultured neuron (1×) with its corresponding axonal growth cone highlighted in a white square box, shown in a confocal section of 0.772 μm in the z -plane at higher (5×) magnification. The negative control for primary antibodies is shown in another growth cone on the right. The image in the bottom is the merge of NP1 and Kv7.2 immunofluorescence images in the single confocal section obtained at 5× showing colocalization (white) of NP1 and Kv7.2 in the growth cone, with the corresponding orthogonal views from its respective stack of confocal sections. Images were acquired using restricted spectral emission wavelength ranges chosen to avoid crosstalk or bleed-through between the three different channels. Scale bar, 5 μm.

Techniques Used: Western Blot, Immunoprecipitation, Molecular Weight, Recombinant, Transfection, Immunofluorescence, Confocal Microscopy, Isolation, Negative Control, Marker, Cell Culture

Knockdown of NP1 reduces Kv7.2 surface expression and M-current. A , B , The proportion of Kv7.2 at the neuronal surface was determined by biotinylation (30 min at 4°C) of all surface proteins in cultured cortical neurons. The ratio of the surface over the total amount of receptor was determined by densitometric analysis and expressed as a percentage of control values. Only samples of biotinylated proteins lacking immunoreactivity to actin, which indicates contamination with intracellular proteins, were used for analyses. A , Representative Western blots of Kv7.2, Na-KATPase, and actin from total (T) and biotinylated (B) proteins. B , Quantitative analysis of the effect of gain and loss of NP1 on the surface/total levels of the Kv7.2 subunit of the voltage-gated Kv7/M K + channels. Values are mean ± SEM of shRdm ( n = 11), shNP1 ( n = 11), and pWPI-NP1 ( n = 5), from at least three independent experiments; * p
Figure Legend Snippet: Knockdown of NP1 reduces Kv7.2 surface expression and M-current. A , B , The proportion of Kv7.2 at the neuronal surface was determined by biotinylation (30 min at 4°C) of all surface proteins in cultured cortical neurons. The ratio of the surface over the total amount of receptor was determined by densitometric analysis and expressed as a percentage of control values. Only samples of biotinylated proteins lacking immunoreactivity to actin, which indicates contamination with intracellular proteins, were used for analyses. A , Representative Western blots of Kv7.2, Na-KATPase, and actin from total (T) and biotinylated (B) proteins. B , Quantitative analysis of the effect of gain and loss of NP1 on the surface/total levels of the Kv7.2 subunit of the voltage-gated Kv7/M K + channels. Values are mean ± SEM of shRdm ( n = 11), shNP1 ( n = 11), and pWPI-NP1 ( n = 5), from at least three independent experiments; * p

Techniques Used: Expressing, Cell Culture, Western Blot

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    Ipsen Group kv7 2
    NP1 interacts and colocalizes with <t>Kv7.2</t> at presynaptic terminals of excitatory synapses and axonal growth cones. A – D , Representative Western blots of immunoprecipitation eluates separated in 6% Tris-Glycine (for high molecular weight proteins) and 4–12% Bis-Tris gels (for low molecular weight proteins). TL, Total protein lysate; Syx, syntaxin. A , Both native Kv7.2 and syntaxin 1A, but not Kv7.3, coprecipitate with NP1 in total brain extracts. B , Both native Kv7.2 and NP1 coprecipitate with syntaxin in total brain extracts. C , D , Recombinant NP1 coprecipitates Kv7.2, but not syntaxin or Kv7.3, in 293T cells transfected with NP1, 5Myc-Kv7.2, 2HA-Kv7.3, and syntaxin 1A cDNAs. Kv7.2 and Kv7.3 were immunoprecipitated with antibodies against their respective Myc and HA tags. E , F , Immunofluorescence studies and confocal microscopy were performed in high-density ( E ) or low-density ( F ) isolated cortical neurons. E , Top, Confocal sections of 0.772 μm in the z -plane showing immunofluorescence of NP1, VGLUT1, Kv7.2, and negative control (omitting primary antibodies). Bottom, Colocalization (in white) of the excitatory presynaptic marker VGLUT1 (blue) with NP1 (green) and Kv7.2 (red) is shown in a single section with the corresponding orthogonal views of the stack of confocal sections. White arrows indicate sites of colocalization. F , NP1 (green) and KV7.2 (magenta) immunofluorescence and DIC images of an isolated cortical cultured neuron (1×) with its corresponding axonal growth cone highlighted in a white square box, shown in a confocal section of 0.772 μm in the z -plane at higher (5×) magnification. The negative control for primary antibodies is shown in another growth cone on the right. The image in the bottom is the merge of NP1 and Kv7.2 immunofluorescence images in the single confocal section obtained at 5× showing colocalization (white) of NP1 and Kv7.2 in the growth cone, with the corresponding orthogonal views from its respective stack of confocal sections. Images were acquired using restricted spectral emission wavelength ranges chosen to avoid crosstalk or bleed-through between the three different channels. Scale bar, 5 μm.
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    NP1 interacts and colocalizes with Kv7.2 at presynaptic terminals of excitatory synapses and axonal growth cones. A – D , Representative Western blots of immunoprecipitation eluates separated in 6% Tris-Glycine (for high molecular weight proteins) and 4–12% Bis-Tris gels (for low molecular weight proteins). TL, Total protein lysate; Syx, syntaxin. A , Both native Kv7.2 and syntaxin 1A, but not Kv7.3, coprecipitate with NP1 in total brain extracts. B , Both native Kv7.2 and NP1 coprecipitate with syntaxin in total brain extracts. C , D , Recombinant NP1 coprecipitates Kv7.2, but not syntaxin or Kv7.3, in 293T cells transfected with NP1, 5Myc-Kv7.2, 2HA-Kv7.3, and syntaxin 1A cDNAs. Kv7.2 and Kv7.3 were immunoprecipitated with antibodies against their respective Myc and HA tags. E , F , Immunofluorescence studies and confocal microscopy were performed in high-density ( E ) or low-density ( F ) isolated cortical neurons. E , Top, Confocal sections of 0.772 μm in the z -plane showing immunofluorescence of NP1, VGLUT1, Kv7.2, and negative control (omitting primary antibodies). Bottom, Colocalization (in white) of the excitatory presynaptic marker VGLUT1 (blue) with NP1 (green) and Kv7.2 (red) is shown in a single section with the corresponding orthogonal views of the stack of confocal sections. White arrows indicate sites of colocalization. F , NP1 (green) and KV7.2 (magenta) immunofluorescence and DIC images of an isolated cortical cultured neuron (1×) with its corresponding axonal growth cone highlighted in a white square box, shown in a confocal section of 0.772 μm in the z -plane at higher (5×) magnification. The negative control for primary antibodies is shown in another growth cone on the right. The image in the bottom is the merge of NP1 and Kv7.2 immunofluorescence images in the single confocal section obtained at 5× showing colocalization (white) of NP1 and Kv7.2 in the growth cone, with the corresponding orthogonal views from its respective stack of confocal sections. Images were acquired using restricted spectral emission wavelength ranges chosen to avoid crosstalk or bleed-through between the three different channels. Scale bar, 5 μm.

    Journal: The Journal of Neuroscience

    Article Title: Neuronal Pentraxin 1 Negatively Regulates Excitatory Synapse Density and Synaptic Plasticity

    doi: 10.1523/JNEUROSCI.2548-14.2015

    Figure Lengend Snippet: NP1 interacts and colocalizes with Kv7.2 at presynaptic terminals of excitatory synapses and axonal growth cones. A – D , Representative Western blots of immunoprecipitation eluates separated in 6% Tris-Glycine (for high molecular weight proteins) and 4–12% Bis-Tris gels (for low molecular weight proteins). TL, Total protein lysate; Syx, syntaxin. A , Both native Kv7.2 and syntaxin 1A, but not Kv7.3, coprecipitate with NP1 in total brain extracts. B , Both native Kv7.2 and NP1 coprecipitate with syntaxin in total brain extracts. C , D , Recombinant NP1 coprecipitates Kv7.2, but not syntaxin or Kv7.3, in 293T cells transfected with NP1, 5Myc-Kv7.2, 2HA-Kv7.3, and syntaxin 1A cDNAs. Kv7.2 and Kv7.3 were immunoprecipitated with antibodies against their respective Myc and HA tags. E , F , Immunofluorescence studies and confocal microscopy were performed in high-density ( E ) or low-density ( F ) isolated cortical neurons. E , Top, Confocal sections of 0.772 μm in the z -plane showing immunofluorescence of NP1, VGLUT1, Kv7.2, and negative control (omitting primary antibodies). Bottom, Colocalization (in white) of the excitatory presynaptic marker VGLUT1 (blue) with NP1 (green) and Kv7.2 (red) is shown in a single section with the corresponding orthogonal views of the stack of confocal sections. White arrows indicate sites of colocalization. F , NP1 (green) and KV7.2 (magenta) immunofluorescence and DIC images of an isolated cortical cultured neuron (1×) with its corresponding axonal growth cone highlighted in a white square box, shown in a confocal section of 0.772 μm in the z -plane at higher (5×) magnification. The negative control for primary antibodies is shown in another growth cone on the right. The image in the bottom is the merge of NP1 and Kv7.2 immunofluorescence images in the single confocal section obtained at 5× showing colocalization (white) of NP1 and Kv7.2 in the growth cone, with the corresponding orthogonal views from its respective stack of confocal sections. Images were acquired using restricted spectral emission wavelength ranges chosen to avoid crosstalk or bleed-through between the three different channels. Scale bar, 5 μm.

    Article Snippet: To further investigate the interaction between NP1 and Kv7.2 and its relationship with synapses in cultured cortical neurons, we performed triple-labeling immunofluorescence experiments and analysis by confocal microscopy ( E , F ).

    Techniques: Western Blot, Immunoprecipitation, Molecular Weight, Recombinant, Transfection, Immunofluorescence, Confocal Microscopy, Isolation, Negative Control, Marker, Cell Culture

    Knockdown of NP1 reduces Kv7.2 surface expression and M-current. A , B , The proportion of Kv7.2 at the neuronal surface was determined by biotinylation (30 min at 4°C) of all surface proteins in cultured cortical neurons. The ratio of the surface over the total amount of receptor was determined by densitometric analysis and expressed as a percentage of control values. Only samples of biotinylated proteins lacking immunoreactivity to actin, which indicates contamination with intracellular proteins, were used for analyses. A , Representative Western blots of Kv7.2, Na-KATPase, and actin from total (T) and biotinylated (B) proteins. B , Quantitative analysis of the effect of gain and loss of NP1 on the surface/total levels of the Kv7.2 subunit of the voltage-gated Kv7/M K + channels. Values are mean ± SEM of shRdm ( n = 11), shNP1 ( n = 11), and pWPI-NP1 ( n = 5), from at least three independent experiments; * p

    Journal: The Journal of Neuroscience

    Article Title: Neuronal Pentraxin 1 Negatively Regulates Excitatory Synapse Density and Synaptic Plasticity

    doi: 10.1523/JNEUROSCI.2548-14.2015

    Figure Lengend Snippet: Knockdown of NP1 reduces Kv7.2 surface expression and M-current. A , B , The proportion of Kv7.2 at the neuronal surface was determined by biotinylation (30 min at 4°C) of all surface proteins in cultured cortical neurons. The ratio of the surface over the total amount of receptor was determined by densitometric analysis and expressed as a percentage of control values. Only samples of biotinylated proteins lacking immunoreactivity to actin, which indicates contamination with intracellular proteins, were used for analyses. A , Representative Western blots of Kv7.2, Na-KATPase, and actin from total (T) and biotinylated (B) proteins. B , Quantitative analysis of the effect of gain and loss of NP1 on the surface/total levels of the Kv7.2 subunit of the voltage-gated Kv7/M K + channels. Values are mean ± SEM of shRdm ( n = 11), shNP1 ( n = 11), and pWPI-NP1 ( n = 5), from at least three independent experiments; * p

    Article Snippet: To further investigate the interaction between NP1 and Kv7.2 and its relationship with synapses in cultured cortical neurons, we performed triple-labeling immunofluorescence experiments and analysis by confocal microscopy ( E , F ).

    Techniques: Expressing, Cell Culture, Western Blot