kv7 2  (Alomone Labs)

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: Incubation with primary antibodies was performed overnight at 4°C with the following antibodies, diluted in 3% BSA/PBS or 1% NGS/PBS (NP1, VGLUT1, and Kv7.2): mouse monoclonal anti-PSD95 (1:50; Abcam), anti-gephyrin (1:100; Synaptic Systems), anti-NP1 (1:50, BD Transduction), rabbit polyclonal anti-synaptophysin (1:100; Synaptic Systems), anti-Kv7.2 (1:100; Alomone Labs), and guinea pig anti-VGLUT1 (1:200; Millipore Bioscience Research Reagents).

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

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: Incubation with primary antibodies was performed overnight at 4°C with the following antibodies, diluted in 3% BSA/PBS or 1% NGS/PBS (NP1, VGLUT1, and Kv7.2): mouse monoclonal anti-PSD95 (1:50; Abcam), anti-gephyrin (1:100; Synaptic Systems), anti-NP1 (1:50, BD Transduction), rabbit polyclonal anti-synaptophysin (1:100; Synaptic Systems), anti-Kv7.2 (1:100; Alomone Labs), and guinea pig anti-VGLUT1 (1:200; Millipore Bioscience Research Reagents).

Techniques: Expressing, Cell Culture, Western Blot

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: We used the following antibodies: mouse anti-NP1 antibody (1:1000; Becton Dickinson), mouse anti-synaptophysin, clone SY38 (1:1000), mouse anti-PSD95 (1:2000; both from Millipore Bioscience Research Reagents), rabbit anti-Kv7.2 (1:200; Alomone Labs), mouse anti-syntaxin 1A (HPC-1 clone, 1:2000; Sigma-Aldrich), mouse anti-HA (1:3000, Covance), and mouse anti-Myc (9E10 clone, 1:5000; Sigma-Aldrich).

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

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: We used the following antibodies: mouse anti-NP1 antibody (1:1000; Becton Dickinson), mouse anti-synaptophysin, clone SY38 (1:1000), mouse anti-PSD95 (1:2000; both from Millipore Bioscience Research Reagents), rabbit anti-Kv7.2 (1:200; Alomone Labs), mouse anti-syntaxin 1A (HPC-1 clone, 1:2000; Sigma-Aldrich), mouse anti-HA (1:3000, Covance), and mouse anti-Myc (9E10 clone, 1:5000; Sigma-Aldrich).

Techniques: Expressing, Cell Culture, Western Blot

Journal: Addiction biology

Article Title: Kv7 Channels in the Nucleus Accumbens are altered by Chronic Drinking and are Targets for Reducing Alcohol Consumption

doi: 10.1111/adb.12279

Figure Lengend Snippet: Long-term drinking reduced SUMOylation levels of Kv7.2 in the NAc. (a) Schematic depicting locations of the SUMOylation consensus sequences in rat Kv7.2 (orange circle and arrow indicate point of SUMO attachment). (b) Representative western blot images showing the expression of SUMOylated Kv7.2 in fractionated NAc tissue. (c) 72 hr withdrawal from alcohol decreased expression of SUMOylated Kv7.2 in the DSM but not the DRM fraction. (d) However, IAA exposure reduced the ratio of SUMOylated Kv7.2 to total Kv7.2 in the DRM fraction (* p

Article Snippet: The antibodies used in this study were Kv7.2 (#APC-050; Alomone Labs; Jerusalem, Israel), AKAP150 (#07-210; EMD Millipore; Billerica, MA), and SGK1 (#S5188; Sigma-Aldrich Co).

Techniques: Western Blot, Expressing

Journal: Addiction biology

Article Title: Kv7 Channels in the Nucleus Accumbens are altered by Chronic Drinking and are Targets for Reducing Alcohol Consumption

doi: 10.1111/adb.12279

Figure Lengend Snippet: Long-term drinking altered Kv7.2 surface trafficking in the NAc. (a) Representative western blot images showing the relative expression patterns of Kv7.2, AKAP150, and SGK1 in the DRM, IC, and DSM fractions in tissue targeting the NAc core. (b) Representative western blot images of Kv7.2, AKAP150, and SGK1 expression in the NAc of alcohol naïve and IAA rats after 72 hr withdrawal from alcohol. (c) Kv7.2 expression is increased in the DRM of IAA rats and decreased in the DSM. There is no effect of alcohol exposure on the expression of (d) AKAP150 in the DRM or (e) SGK1 in the IC fraction (* p

Article Snippet: The antibodies used in this study were Kv7.2 (#APC-050; Alomone Labs; Jerusalem, Israel), AKAP150 (#07-210; EMD Millipore; Billerica, MA), and SGK1 (#S5188; Sigma-Aldrich Co).

Techniques: Western Blot, Expressing

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: The bound biotinylated proteins were eluted by incubating for 1 h at room temperature with SDS-PAGE buffer (62.5 m m Tris-HCl, pH 6.8, 1% SDS, 10% glycerol, and 50 m m DTT) followed by centrifugation for 2 min at 1000 × g. Biotinylated proteins were separated in 8% SDS-PAGE and transferred to PVDF membranes (Millipore), which were probed with one of the following antibodies: rabbit anti-Kv7.2 antibody (1:2000; Alomone Labs), rabbit anti-GluA1 antibody (1:1000; Millipore), mouse monoclonal anti-GluA2 antibody (1:1000; Neuromab), and mouse monoclonal anti-GluN1 (1:2000; Becton Dickinson) as described above.

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

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: The bound biotinylated proteins were eluted by incubating for 1 h at room temperature with SDS-PAGE buffer (62.5 m m Tris-HCl, pH 6.8, 1% SDS, 10% glycerol, and 50 m m DTT) followed by centrifugation for 2 min at 1000 × g. Biotinylated proteins were separated in 8% SDS-PAGE and transferred to PVDF membranes (Millipore), which were probed with one of the following antibodies: rabbit anti-Kv7.2 antibody (1:2000; Alomone Labs), rabbit anti-GluA1 antibody (1:1000; Millipore), mouse monoclonal anti-GluA2 antibody (1:1000; Neuromab), and mouse monoclonal anti-GluN1 (1:2000; Becton Dickinson) as described above.

Techniques: Expressing, Cell Culture, Western Blot