Journal: The Journal of Biological Chemistry

Article Title: γ-Aminobutyric Acid Type A (GABA A ) Receptor Subunits Play a Direct Structural Role in Synaptic Contact Formation via Their N-terminal Extracellular Domains *

doi: 10.1074/jbc.M116.714790

Figure Lengend Snippet: Formation of synapse-like contacts between GABAergic MSNs and HEK293 cells expressing α1/β2/γ2-GABA A Rs at the cell surface. A, immunoblot analysis of the expression of GABA A R α1 ( left panels ) and β2 ( right panels ) subunits in a stable α1β2-HEK293 cell line in comparison with the normal HEK293 cells, using specific primary antibodies and alkaline phosphatase-conjugated secondary antibodies for detection. Representative blots of n = 2 independent experiments are shown. B, immunocytochemical analysis of the expression of GABA A R α1 ( left panel ) and β2 ( middle panel ) subunits in a stable α1β2-HEK293 cell line, using primary antibodies specifically binding to the extracellular epitopes of the respective subunits and Alexa488-coupled ( left panel ) or Alexa555-coupled ( middle panel ) secondary antibodies. Differential interference contrast ( DIC ) image of the same field ( right panel ) is included for comparison. Scale bar, 10 μm. Representative images of n = 2 independent experiments are shown. C, immunolabeling of synaptic contacts between GABAergic presynaptic terminals of MSNs expressing VGAT and α1β2γ2-HEK293 cells ( top panel ), α1β2-HEK293 cells ( middle panel ), or HEK293 cells ( bottom panel ). GABAergic terminals were labeled with an anti-VGAT antibody (in green ); HEK293 cells were labeled with mCherry (in red ), and the surface-expressed GABA A Rs were labeled with the γ2 subunit-specific antibody (in blue ). A selected area in each image ( left column, white box ) was magnified four times, and these images were included in the right column. Scale bar, 10 μm. D, quantification of contacts between MSNs and α1β2γ2-HEK293 cells, α1β2-HEK293 cells, or control HEK293 cells expressed as a percentage of co-localized pixels between VGAT-positive MSN terminals and mCherry-expressing HEK293 cells. The data were first analyzed using Shapiro-Wilk and Kolmogorov-Smirnov tests and subsequently using the non-parametric Mann-Whitney test with a confidence interval of 95%. The box plots display the median and interquartile range ( IQR ); small squares represent the mean, and whiskers represent the data range within 1 S.D. of the median ( n = 62–77 cells from n = 6 independent experiments). E, cell surface levels of GABA A R β2 subunit in α1β2-HEK293 cells showed no significant change following transient transfection of the γ2 subunit (α1β2γ2-HEK293 cells), as measured using ELISA with an extracellular epitope-specific anti-β2/3 subunit antibody, followed by an HRP-conjugated secondary antibody and colorimetric reaction ( left panel ; p > 0.05, paired t test). Total levels of GABA A R β2 subunit in α1β2γ2-HEK293 cells were reduced ( right panel ; p < 0.05, paired t test). The bar graphs represent the mean ± S.D. of the total of n = 4 independent experiments. The superimposed paired scatterplots represent the individual data points obtained from α1β2-HEK293 and α1β2γ2-HEK293 cells. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: Samples were separated using SDS-PAGE and transferred onto a nitrocellulose membrane, which was then incubated with His 6 tag antibody (1:1000; catalog no. AM1010a, Abgent) or the subunit-specific primary antibodies as follows: rabbit anti-GABA A R γ2 subunit (1:1000; catalog no. 224003, Synaptic Systems); anti-GABA A R β2/β3 subunit (1:50; UCL 112, raised against the N-terminal peptide of the β2 subunit in Dr. J. Jovanovic's laboratory); anti-GABA A R β3 subunit (1:500, UCL 74 ( )); anti-GABA A R α1 subunit (1:500 ( )); anti-GABA A R α2 subunit (1:500, catalog no. 224102, Synaptic Systems), or anti-GABA A R α2 subunit (2 μg/μl, raised against the intracellular loop (416–424) of the α2 subunit ( )).

Techniques: Expressing, Western Blot, Binding Assay, Immunolabeling, Labeling, MANN-WHITNEY, Transfection, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Biological Chemistry

Article Title: γ-Aminobutyric Acid Type A (GABA A ) Receptor Subunits Play a Direct Structural Role in Synaptic Contact Formation via Their N-terminal Extracellular Domains *

doi: 10.1074/jbc.M116.714790

Figure Lengend Snippet: Formation of synapse-like contacts between GABAergic MSNs and HEK293 cells expressing α2/β2/γ2-GABA A Rs at the cell surface. A, immunoblot analysis of the expression of GABA A R α2 ( left panel ) and β2 ( right panel ) subunits in a stable α2β2-HEK293 cell line in comparison with the normal HEK293 cells, using specific primary antibodies and alkaline phosphatase-conjugated secondary antibodies for detection. Representative blots of n = 2 independent experiments are shown. B, immunocytochemical analysis of the expression of GABA A R α2 ( left panel ) and β2 ( middle panel ) subunits in a stable α2β2-HEK293 cell line, using primary antibodies specifically binding to the extracellular epitopes of the respective subunits and Alexa488-coupled ( left panel ) or Alexa555-coupled ( middle panel ) secondary antibodies. DIC image of the same field ( right panel ) is included for comparison. Scale bar, 10 μm. Representative images of n = 2 independent experiments are shown. C, immunolabeling of synaptic contacts between GABAergic presynaptic terminals of MSNs expressing VGAT and α2β2γ2-HEK293 cells ( top panel ), α2β2-HEK293 cells ( middle panel ), or HEK293 cells ( bottom panel ). GABAergic terminals were labeled with an anti-VGAT antibody (in green ); HEK293 cells were labeled with mCherry (in red ), and the surface-expressed GABA A Rs were labeled with the γ2 subunit-specific antibody (in blue ). A selected area in each image ( left column, white box ) was magnified four times, and these images are included in the right column. Scale bar, 10 μm. D, quantification of contacts between MSNs and α2β2γ2-HEK293 cells, α2β2-HEK293 cells or control HEK293 cells expressed as a percentage of co-localized pixels between VGAT positive MSN terminals and mCherry expressing HEK293 cells. The data were first analyzed using Shapiro-Wilk and Kolmogorov-Smirnov tests and subsequently using the non-parametric Mann-Whitney test with a confidence interval of 95%. The box plots display the median and IQR; small squares represent the mean, and whiskers represent the data range within 1 S.D. of the median ( n = 42–56 cells from n = 3 independent experiments). E, cell surface levels of GABA A R β2 subunit in α2β2-HEK293 cells showed no significant change following transient transfection of the γ2 subunit (α2β2γ2-HEK293 cells), as measured using ELISA with an extracellular epitope-specific anti-β2/3 subunit antibody, followed by an HRP-conjugated secondary antibody and colorimetric reaction ( left panel ; p > 0.05, paired t test). Total levels of GABA A R β2 subunit in α2β2γ2-HEK293 cells were reduced ( right panel ; p < 0.05, paired t test). The bar graphs represent the mean ± S.D. of the total of n = 3 (for cell surface expression) and n = 4 (for total expression) independent experiments. The superimposed paired scatterplots represent the individual data points obtained from α2β2-HEK293 and α2β2γ2-HEK293 cells. *, p < 0.05; **, p < 0.01.

Article Snippet: Samples were separated using SDS-PAGE and transferred onto a nitrocellulose membrane, which was then incubated with His 6 tag antibody (1:1000; catalog no. AM1010a, Abgent) or the subunit-specific primary antibodies as follows: rabbit anti-GABA A R γ2 subunit (1:1000; catalog no. 224003, Synaptic Systems); anti-GABA A R β2/β3 subunit (1:50; UCL 112, raised against the N-terminal peptide of the β2 subunit in Dr. J. Jovanovic's laboratory); anti-GABA A R β3 subunit (1:500, UCL 74 ( )); anti-GABA A R α1 subunit (1:500 ( )); anti-GABA A R α2 subunit (1:500, catalog no. 224102, Synaptic Systems), or anti-GABA A R α2 subunit (2 μg/μl, raised against the intracellular loop (416–424) of the α2 subunit ( )).

Techniques: Expressing, Western Blot, Binding Assay, Immunolabeling, Labeling, MANN-WHITNEY, Transfection, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Biological Chemistry

Article Title: γ-Aminobutyric Acid Type A (GABA A ) Receptor Subunits Play a Direct Structural Role in Synaptic Contact Formation via Their N-terminal Extracellular Domains *

doi: 10.1074/jbc.M116.714790

Figure Lengend Snippet: Formation of synapse-like contacts between GABAergic MSNs and HEK293 cells expressing α1/β3/γ2-GABA A Rs at the cell surface. A, immunoblot analysis of the expression of GABA A R α1 ( left panel ) and β3 ( right panel ) subunits in a stable α1β3-HEK293 cell line in comparison with the normal HEK293 cells, using specific primary antibodies and alkaline phosphatase-conjugated secondary antibodies for detection. Representative blots of n = 2 independent experiments are shown. B, immunocytochemical analysis of the expression of GABA A R α1 ( left panel ) and β3 ( middle panel ) subunits in a stable α1β3-HEK293 cell line, using primary antibodies specifically binding to the extracellular epitopes of the respective subunits and Alexa488-coupled ( left panel ) or Alexa555-coupled ( middle panel ) secondary antibodies. DIC image of the same field ( right panel ) is included for comparison. Scale bar, 10 μm. Representative images of n = 2 independent experiments are shown. C, immunolabeling of synaptic contacts between GABAergic presynaptic terminals of MSNs expressing VGAT and α1β3γ2-HEK293 cells ( top panel ), α1β3-HEK293 cells ( middle panel ), or HEK293 cells ( bottom panel ). GABAergic terminals were labeled with an anti-VGAT antibody (in green ); HEK293 cells were labeled with mCherry (in red ); and the surface-expressed GABA A Rs were labeled with the γ2 subunit-specific antibody (in blue ). A selected area in each image ( left column, white box ) was magnified four times, and these images were included in the right column. Scale bar, 10 μm. D, quantification of contacts between MSNs and α1β3γ2-HEK293 cells, α1β3-HEK293 cells or control HEK293 cells expressed as a percentage of co-localized pixels between VGAT-positive MSN terminals and mCherry-expressing HEK293 cells. The data were first analyzed using Shapiro-Wilk and Kolmogorov-Smirnov tests and subsequently using the non-parametric Mann-Whitney test with a confidence interval of 95%. The box plots display the median and IQR; small squares represent the mean, and whiskers represent the data range within 1 S.D. of the median ( n = 37–49 cells from n = 4 independent experiments). E, cell surface levels of GABA A R β3 subunit in α1β3-HEK293 cells showed no significant change following transient transfection of the γ2 subunit (α1β3γ2-HEK293 cells), as measured using ELISA with an extracellular epitope-specific anti-β2/3 subunit antibody, followed by an HRP-conjugated secondary antibody and colorimetric reaction ( left panel ; p > 0.05, paired t test). The bar graphs represent the mean ± S.D. of the total of n = 4 independent experiments. The superimposed paired scatterplots represent the individual data points obtained from α1β3-HEK293 and α1β3γ2-HEK293 cells. ***, p < 0.001.

Article Snippet: Samples were separated using SDS-PAGE and transferred onto a nitrocellulose membrane, which was then incubated with His 6 tag antibody (1:1000; catalog no. AM1010a, Abgent) or the subunit-specific primary antibodies as follows: rabbit anti-GABA A R γ2 subunit (1:1000; catalog no. 224003, Synaptic Systems); anti-GABA A R β2/β3 subunit (1:50; UCL 112, raised against the N-terminal peptide of the β2 subunit in Dr. J. Jovanovic's laboratory); anti-GABA A R β3 subunit (1:500, UCL 74 ( )); anti-GABA A R α1 subunit (1:500 ( )); anti-GABA A R α2 subunit (1:500, catalog no. 224102, Synaptic Systems), or anti-GABA A R α2 subunit (2 μg/μl, raised against the intracellular loop (416–424) of the α2 subunit ( )).

Techniques: Expressing, Western Blot, Binding Assay, Immunolabeling, Labeling, MANN-WHITNEY, Transfection, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Biological Chemistry

Article Title: γ-Aminobutyric Acid Type A (GABA A ) Receptor Subunits Play a Direct Structural Role in Synaptic Contact Formation via Their N-terminal Extracellular Domains *

doi: 10.1074/jbc.M116.714790

Figure Lengend Snippet: Synapse-like contacts do not form between GABAergic MSNs and HEK293 cells expressing α2/β3/γ2-GABA A Rs at the cell surface. A, immunoblot analysis of the expression of GABA A R α2 ( left panel ) and β3 ( right panel ) subunits in a stable α2β3-HEK293 cell line in comparison with the normal HEK293 cells, using specific primary antibodies and alkaline phosphatase-conjugated secondary antibodies for detection. Representative blots of n = 2 independent experiments are shown. B, immunocytochemical analysis of the expression of GABA A R α2 ( left panel ) and β3 ( middle panel ) subunits in a stable α2β3-HEK293 cell line, using primary antibodies specifically binding to the extracellular epitopes of the respective subunits and Alexa488- ( left panel or Alexa555-coupled ( middle panel )) secondary antibodies. DIC image of the same field ( right panel ) is included for comparison. Scale bar , 10 μm. Representative images of n = 2 independent experiments are shown. C, immunolabeling of synaptic contacts between GABAergic presynaptic terminals of MSNs expressing VGAT and α2β3γ2-HEK293 cells ( top panel ), α2β3-HEK293 cells ( middle panel ), or HEK293 cells ( bottom panel ). GABAergic terminals were labeled with an anti-VGAT antibody (in green ); HEK293 cells were labeled with mCherry (in red ), and the surface expressed GABA A Rs were labeled with the γ2 subunit-specific antibody (in blue ). A selected area in each image ( left column, white box ) was magnified four times, and these images were included in the right column. Scale bar, 10 μm. D, quantification of contacts between MSNs and α2β3γ2-HEK293 cells, α2β3-HEK293 cells, or control HEK293 cells expressed as a percentage of co-localized pixels between VGAT-positive MSN terminals and mCherry-expressing HEK293 cells. The data were first analyzed using Shapiro-Wilk and Kolmogorov-Smirnov tests and subsequently using the non-parametric Mann-Whitney test with a confidence interval of 95%. The box plots display the median and IQR; small squares represent the mean, and whiskers represent the data range within 1 S.D. of the median ( n = 42–56 cells from n = 3 independent experiments). E, cell surface levels of GABA A R β3 subunit in α2β3-HEK293 cells showed no significant change following transient transfection of the γ2 subunit (α2β3γ2-HEK293 cells), as measured using ELISA with an extracellular epitope-specific anti-β2/3 subunit antibody, followed by an HRP-conjugated secondary antibody and colorimetric reaction ( left panel ; p > 0.05, paired t test). The bar graphs represent the mean ± S.D. of the total of n = 4 independent experiments. The superimposed paired scatterplots represent the individual data points obtained from α2β3-HEK293 and α2β3γ2-HEK293 cells.

Article Snippet: Samples were separated using SDS-PAGE and transferred onto a nitrocellulose membrane, which was then incubated with His 6 tag antibody (1:1000; catalog no. AM1010a, Abgent) or the subunit-specific primary antibodies as follows: rabbit anti-GABA A R γ2 subunit (1:1000; catalog no. 224003, Synaptic Systems); anti-GABA A R β2/β3 subunit (1:50; UCL 112, raised against the N-terminal peptide of the β2 subunit in Dr. J. Jovanovic's laboratory); anti-GABA A R β3 subunit (1:500, UCL 74 ( )); anti-GABA A R α1 subunit (1:500 ( )); anti-GABA A R α2 subunit (1:500, catalog no. 224102, Synaptic Systems), or anti-GABA A R α2 subunit (2 μg/μl, raised against the intracellular loop (416–424) of the α2 subunit ( )).

Techniques: Expressing, Western Blot, Binding Assay, Immunolabeling, Labeling, MANN-WHITNEY, Transfection, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Biological Chemistry

Article Title: γ-Aminobutyric Acid Type A (GABA A ) Receptor Subunits Play a Direct Structural Role in Synaptic Contact Formation via Their N-terminal Extracellular Domains *

doi: 10.1074/jbc.M116.714790

Figure Lengend Snippet: Inhibition of GABA A R activity by application of bicuculline has no effect on the formation of synapse-like contacts between MSNs and α1β2γ2-HEK293 cells. A, immunolabeling of synaptic contacts between the presynaptic terminals of MSNs and α1β2γ2-HEK293 cells in co-cultures treated with DMSO ( upper panel ) or bicuculline ( lower panel ). GABAergic terminals were labeled with an anti-VGAT antibody (in green ); HEK293 cells were labeled with mCherry (in red ), and the surface-expressed GABA A Rs were labeled with the γ2 subunit-specific antibody (in blue ). A selected area in each image ( left column, white box ) was magnified four times, and these images were included in the right column. Scale bar, 10 μm. B, quantification of contacts between MSNs and α1β2γ2-HEK293 cells expressed as percentage of co-localized pixels between VGAT-positive terminals and mCherry expressing HEK293 cells. The Gaussian distribution of the data was initially tested using Shapiro-Wilk and Kolmogorov-Smirnov tests. The data were subsequently analyzed using the non-parametric Mann-Whitney test with a confidence interval of 95%. The box plots display the median and IQR; small squares represent the mean, and whiskers represent the data range within 1 S.D. of the median of ( n = 38–40 cells from three independent experiments).

Article Snippet: Samples were separated using SDS-PAGE and transferred onto a nitrocellulose membrane, which was then incubated with His 6 tag antibody (1:1000; catalog no. AM1010a, Abgent) or the subunit-specific primary antibodies as follows: rabbit anti-GABA A R γ2 subunit (1:1000; catalog no. 224003, Synaptic Systems); anti-GABA A R β2/β3 subunit (1:50; UCL 112, raised against the N-terminal peptide of the β2 subunit in Dr. J. Jovanovic's laboratory); anti-GABA A R β3 subunit (1:500, UCL 74 ( )); anti-GABA A R α1 subunit (1:500 ( )); anti-GABA A R α2 subunit (1:500, catalog no. 224102, Synaptic Systems), or anti-GABA A R α2 subunit (2 μg/μl, raised against the intracellular loop (416–424) of the α2 subunit ( )).

Techniques: Inhibition, Activity Assay, Immunolabeling, Labeling, Expressing, MANN-WHITNEY

Journal: The Journal of Biological Chemistry

Article Title: γ-Aminobutyric Acid Type A (GABA A ) Receptor Subunits Play a Direct Structural Role in Synaptic Contact Formation via Their N-terminal Extracellular Domains *

doi: 10.1074/jbc.M116.714790

Figure Lengend Snippet: Purification of N-terminal ECDs of GABA A R subunits using affinity chromatography. A, samples were collected throughout the purification of N-terminal ECDs of GABA A R α1 ( panel i ), α2 ( panel ii ), β2 ( panel iii ), and γ2 subunit ( panel iv ) from baculovirus-infected Sf9 cells extracts or control Sf9 cell extracts ( panel v ) and analyzed by SDS-PAGE and immunoblotting using an anti-His 6 tag antibody and alkaline phosphatase-conjugated secondary antibodies. From left to right , input lane , washes 1–5 , elutions 1–3, and beads after elution. B, purified N-terminal ECDs (5 ug) of α1 ( panel i ), α2 ( panel ii ), β2 ( panel iii ), or γ2 subunit ( panel iv ) were analyzed by immunoblotting using subunit-specific antibodies and HRP-conjugated secondary antibodies. C, purified N-terminal ECDs of α1, α2, β2, and γ2 subunits were incubated with deglycosylation enzymes and analyzed by immunoblotting with anti-His 6 tag antibody and alkaline phosphatase-conjugated secondary antibodies.

Article Snippet: Samples were separated using SDS-PAGE and transferred onto a nitrocellulose membrane, which was then incubated with His 6 tag antibody (1:1000; catalog no. AM1010a, Abgent) or the subunit-specific primary antibodies as follows: rabbit anti-GABA A R γ2 subunit (1:1000; catalog no. 224003, Synaptic Systems); anti-GABA A R β2/β3 subunit (1:50; UCL 112, raised against the N-terminal peptide of the β2 subunit in Dr. J. Jovanovic's laboratory); anti-GABA A R β3 subunit (1:500, UCL 74 ( )); anti-GABA A R α1 subunit (1:500 ( )); anti-GABA A R α2 subunit (1:500, catalog no. 224102, Synaptic Systems), or anti-GABA A R α2 subunit (2 μg/μl, raised against the intracellular loop (416–424) of the α2 subunit ( )).

Techniques: Purification, Affinity Chromatography, Infection, SDS Page, Western Blot, Incubation