rabbit polyclonal anti α1 antibody  (R&D Systems)

Journal: eLife

Article Title: Hsp47 promotes biogenesis of multi-subunit neuroreceptors in the endoplasmic reticulum

doi: 10.7554/eLife.84798

Figure Lengend Snippet:

Article Snippet: The rabbit polyclonal anti-GABA A α1 subunit antibody (catalog #: 224203) and rabbit polyclonal anti-GABA A γ2 antibody (catalog #: 224003) were obtained from Synaptic Systems.

Techniques: Transfection, Construct, Control, Recombinant, Plasmid Preparation, Cloning, Software

Journal: eLife

Article Title: Hsp47 promotes biogenesis of multi-subunit neuroreceptors in the endoplasmic reticulum

doi: 10.7554/eLife.84798

Figure Lengend Snippet:

Article Snippet: The rabbit polyclonal anti-GABA A α1 antibody (catalog #: PPS022) was purchased from R&D systems (Minneapolis, MN).

Techniques: Transfection, Construct, Control, Recombinant, Plasmid Preparation, Clone Assay, Software

Journal: eLife

Article Title: Hsp47 promotes biogenesis of multi-subunit neuroreceptors in the endoplasmic reticulum

doi: 10.7554/eLife.84798

Figure Lengend Snippet: Individual GABA A receptor subunits fold in the endoplasmic reticulum (ER). Properly folded subunits assemble into a heteropentamer in the ER for subsequent trafficking to the plasma membrane. Unassembled and misfolded subunits are degraded by the ER-associated degradation (ERAD) pathway. The GABA A receptor cartoons are built from the cryo-EM structure of human α1β2γ2 GABA A receptors (PDB: 6X3 S). The large intracellular loop between TM3-TM4 is missing in the structure.

Article Snippet: Antibody , anti-GABA A α1 (rabbit polyclonal) , Synaptic Systems , Cat#: 224203 , IF (1:250).

Techniques: Membrane, Cryo-EM Sample Prep

Journal: eLife

Article Title: Hsp47 promotes biogenesis of multi-subunit neuroreceptors in the endoplasmic reticulum

doi: 10.7554/eLife.84798

Figure Lengend Snippet: ( A ) Endogenous interactions between GABA A receptor α1 subunits and Hsp47. Mouse brain homogenates from 8 to 10 weeks C57BL/6 J mice were immunoprecipitated with an anti-α1 antibody, and the immunoisolated eluents were blotted with indicated antibodies. IgG was included as a negative control for non-specific binding. Three biological replicates were performed. ( B ) Recombinant Hsp47 binds recombinant α1 subunit and β2 subunit of GABA A receptors in vitro. GST, GST-tagged α1 or GST-tagged β2 recombinant protein was mixed with His-tagged Hsp47 in buffers containing 1% Triton X-100. The protein complex was isolated by immunoprecipitation using an anti-His antibody, and the immunopurified eluents were separated by SDS-PAGE and blotted with indicated antibodies. Three biological replicates were performed. ( C ) MicroScale Thermophoresis (MST) was used to determine the binding affinities between Hsp47, an ER luminal chaperone, to RED-labeled His-α1(ERD) and His-β2(ERD). Increasing concentrations of recombinant Hsp47 proteins (0.2 nM – 10 μM) were incubated with 50 nM RED-labeled His-α1(ERD) or His-β2(ERD) in PBS with Tween-20 (0.05%). Then samples were loaded to the capillaries and measured using a Monolith NT.115 instrument with the settings of 40% LED/excitation and 40% MST power. Three biological replicates were performed. The data were analyzed using the Monolith software for the calculation of the dissociation constant (Kd). IP, immunoprecipitation; IB, immunoblotting. Figure 1—source data 1. Original files for the western blot analysis in . Figure 1—source data 2. PDF containing the original blots in with the relevant bands clearly labeled.

Article Snippet: Antibody , anti-GABA A α1 (rabbit polyclonal) , Synaptic Systems , Cat#: 224203 , IF (1:250).

Techniques: Immunoprecipitation, Negative Control, Binding Assay, Recombinant, In Vitro, Isolation, SDS Page, Microscale Thermophoresis, Labeling, Incubation, Software, Western Blot

Journal: eLife

Article Title: Hsp47 promotes biogenesis of multi-subunit neuroreceptors in the endoplasmic reticulum

doi: 10.7554/eLife.84798

Figure Lengend Snippet: ( A ) Recombinant His-tagged Hsp47 protein was mixed with FLAG-tagged ZIP7, hERG, GST-tagged GABA A receptor α1 recombinant proteins, or buffer only in binding buffers (50 mM Tris, pH 7.5, 150 mM NaCl, and 2 mM N-dodecyl-β-D-maltoside (DDM)). The protein complex was isolated by immunoprecipitation using an anti-His antibody, and the immunopurified eluents were separated by SDS-PAGE and blotted with indicated antibodies. Three biological replicates were performed. ( B ) Representative circular dichroism (CD) spectra of α1 subunit ERD domain and β2 subunit ERD domain. Molar ellipticity [θ] was plotted against the wavelength (nm). Each CD Spectrum was measured by accumulating three spectra to obtain the average with the blank correction. Figure 1—figure supplement 2—source data 1. Original files for the western blot analysis in . Figure 1—figure supplement 2—source data 2. PDF containing the original blots in with the relevant bands clearly labeled.

Article Snippet: Antibody , anti-GABA A α1 (rabbit polyclonal) , Synaptic Systems , Cat#: 224203 , IF (1:250).

Techniques: Recombinant, Binding Assay, Isolation, Immunoprecipitation, SDS Page, Circular Dichroism, Western Blot, Labeling

Journal: eLife

Article Title: Hsp47 promotes biogenesis of multi-subunit neuroreceptors in the endoplasmic reticulum

doi: 10.7554/eLife.84798

Figure Lengend Snippet: ( A ) Hsp47 and GABA A receptor β2/β3 subunit protein expression in various mouse brain regions according to SDS-PAGE and Western blot analysis. Three biological replicate experiments were performed from tissue isolated from three different mice for each brain region. ( B ) Hsp47 knockdown in cultured rat hippocampal neurons. Cultured neurons were subjected to transduction with SERPINH1 siRNA lentivirus or scrambled siRNA lentivirus at days in vitro (DIV) 10. Forty-eight hours post-transduction, neurons were fixed, permeabilized, and stained using anti-Hsp47 or anti-NeuN (a marker of the neuron nuclei) antibodies. Neurons were visualized using a confocal microscope. Representative images are shown for each condition. Scale bar = 15 μm. in the bottom panel, we display the quantification of the Hsp47 staining fluorescence intensity after background correction. The analysis was performed on at least 20 cells accumulated from a minimum of three individual coverslips from either the SERPINH1 siRNA lentivirus or scrambled siRNA lentivirus conditions. Each data point is reported as mean ± SD. Statistical significance was calculated using an unpaired two-tailed Student’s t-Test. *** p<0.001. Figure 2—figure supplement 1—source data 1. Original files for the western blot analysis in . Figure 2—figure supplement 1—source data 2. PDF containing the original blots in with the relevant bands clearly labeled. Figure 2—figure supplement 1—source data 3. Data used for graphs presented in .

Article Snippet: Antibody , anti-GABA A α1 (rabbit polyclonal) , Synaptic Systems , Cat#: 224203 , IF (1:250).

Techniques: Expressing, SDS Page, Western Blot, Isolation, Knockdown, Cell Culture, Transduction, In Vitro, Staining, Marker, Microscopy, Fluorescence, Two Tailed Test, Labeling

Journal: eLife

Article Title: Hsp47 promotes biogenesis of multi-subunit neuroreceptors in the endoplasmic reticulum

doi: 10.7554/eLife.84798

Figure Lengend Snippet: ( A, B ) Effect of knocking down Hsp47 ( A ) and overexpressing Hsp47 ( B ) on the surface expression of endogenous GABA A receptor subunits in primary rat hippocampal neurons. Cultured neurons were transduced with SERPINH1 siRNA lentivirus or scrambled siRNA lentivirus ( A ) and with SERPINH1 cDNA lentivirus or empty vector (EV) lentivirus ( B ) at days in vitro (DIV) 10. Forty-eight hours post transduction, surface GABA A receptors were stained using anti-α1 subunit, anti-β2/β3 subunit, or anti-γ2 subunit antibodies without membrane permeabilization. The cells were then washed, and permeabilized before we stained the nuclei with DAPI. Hsp47 staining was carried out after membrane permeabilization. At least 20 neurons from at least three transductions were imaged by confocal microscopy for each condition. Representative images are shown on the left side. Scale bar = 10 μm ( A ) or 20 μm ( B ). Quantification of the fluorescence intensity of the surface GABA A receptor subunits or Hsp47 after background correction per neuron was shown on the right. ( C ) Whole-cell patch clamping was performed to record GABA-induced currents. Neurons were subjected to transduction as in ( A ) and ( B ). The recordings were carried out 48 hr post transduction. Eight to ten neurons from three transductions were recorded. Representative traces are shown in the left-hand panel. Peak current amplitude ( I max ) is shown on the right. The holding potential was set at −60 mV. pA: picoampere. Each data point is reported as mean ± SD. Statistical significance was calculated using t-test ( A, B ) or one-way ANOVA followed by post hoc Tukey’s HSD test ( C ). *** p<0.001. Figure 2—source data 1. Data used for graphs presented in .

Article Snippet: Antibody , anti-GABA A α1 (rabbit polyclonal) , Synaptic Systems , Cat#: 224203 , IF (1:250).

Techniques: Expressing, Cell Culture, Transduction, Plasmid Preparation, In Vitro, Staining, Membrane, Confocal Microscopy, Fluorescence

Journal: eLife

Article Title: Hsp47 promotes biogenesis of multi-subunit neuroreceptors in the endoplasmic reticulum

doi: 10.7554/eLife.84798

Figure Lengend Snippet: ( A ) Overexpression of Hsp47 increases the endo H-resistant post-ER glycoform of the α1 subunit in HEK293T cells stably expressing α1β2γ2 GABA A receptors. The peptide-N-glycosidase F (PNGase F) enzyme cleaves the innermost GlcNAc and serves a control for unglycosylated α1 proteins (lane 5). Two endo H-resistant bands were detected for the α1 subunit since there are two N-glycosylation sites in α1, indicated by the bracket (lanes 2 and 4). Quantification of the ratio of endo H-resistant / total α1 subunit bands, as a measure of the ER-to-Golgi trafficking efficiency, is shown on the bottom. ( B ) Dithiothreitol (DTT) treatment decreases the interaction between Hsp47 and α1 subunit of GABA A receptors. HEK293T cells stably expressing WT α1β2γ2 GABA A receptors were treated with indicated concentration of DTT in the PBS buffer for 10 min. Then Triton X-100 cell extracts were immunoprecipitated with a mouse anti-Hsp47 antibody, and the immunoisolated eluents were subjected for immunoblotting assay. Quantification of the relative intensity of α1/Hsp47 post IP, as a measure of their interactions, is shown on the bottom panel. ( C ) Disulfide bond mutations in the α1 subunit decrease the interaction between Hsp47 and α1 subunit of GABA A receptors. HEK293T cells were transiently transfected with WT α1β2γ2, α1(C166A)β2γ2, or α1(C166A, C180A)β2γ2 subunits. Forty-eight hours post transfection, Triton X-100 cell extracts were immunoprecipitated with a mouse anti-Hsp47 antibody, and the immunoisolated eluents were subjected for immunoblotting assay. Quantification of the relative intensity of α1/Hsp47 post IP is shown on the bottom panel. ( D ) Disulfide bond mutations in the α1 subunits decrease the solubility of the α1 subunit protein. HEK293T cells were transiently transfected as in ( C ). Forty-eight hours post transfection, the Triton X-100 detergent soluble fractions and the Triton X-100 detergent insoluble fractions were isolated for immunoblotting assay. Quantification of the ratio of insoluble/soluble fractions, as a measure of relative aggregation, is shown on the bottom panel. ( E ) DTT treatment increases the interaction between BiP and α1 subunit of GABA A receptors. HEK293T cells stably expressing α1β2γ2 GABA A receptors were treated with indicated concentrations of DTT in PBS for 10 minutes. Then Triton X-100 cell extracts were immunoprecipitated with a mouse anti-α1 antibody, and the immunoisolated eluents were subjected for immunoblotting assay. Quantification of the relative intensity of BiP/α1 post IP is shown on the bottom panel. ( F ) The disulfide mutations of α1 subunit increase the interaction between BiP and the α1 subunit. HEK293T cells were transiently transfected as in ( C ). Forty-eight hours post transfection, Triton X-100 cell extracts were immunoprecipitated with a mouse anti-α1 antibody, and the immunoisolated eluents were subjected for immunoblotting assay. Quantification of the relative intensity of BiP/α1 post IP is shown on the bottom panel. IP, immunoprecipitation; IB, immunoblotting. For ( A )-( F ), three biological replicates were performed. Each data point is reported as mean ± SD. Significant difference was analyzed by t-test ( A ), or a one-way ANOVA followed by post hoc Tukey’s HSD test ( B–F ). *, p<0.05; **, p<0.01; ***, p<0.001. Figure 3—source data 1. Original files for the western blot analysis in . Figure 3—source data 2. PDF containing the original blots in with the relevant bands clearly labeled. Figure 3—source data 3. Data used for graphs presented in .

Article Snippet: Antibody , anti-GABA A α1 (rabbit polyclonal) , Synaptic Systems , Cat#: 224203 , IF (1:250).

Techniques: Over Expression, Stable Transfection, Expressing, Control, Concentration Assay, Immunoprecipitation, Western Blot, Transfection, Solubility, Isolation, Labeling

Journal: eLife

Article Title: Hsp47 promotes biogenesis of multi-subunit neuroreceptors in the endoplasmic reticulum

doi: 10.7554/eLife.84798

Figure Lengend Snippet: ( A ) Whole-cell patch clamping was performed to record GABA-induced currents. HEK293T cells expressing α1β2γ2 GABA A receptors were transfected with empty vector (EV) control or SERPINH1 cDNA plasmids. The recording was carried out 48 hr post transfection. The holding potential was set at –60 mV. Representative traces were shown. Quantification of the peak currents ( I max ) from 11 to 12 cells from three transfections is shown on the right. pA: picoampere. ( B ) HEK293T cells expressing α1β2γ2 GABA A receptors were transfected with HA-ubiquitin together with empty vector (EV) control or SERPINH1 cDNA plasmids. Forty-eight hours post transfection, cells were lysed and the total proteins were immunoprecipitated with anti-α1 antibody. The eluents were probed with indicated antibodies. Three biological replicates were performed. ( C, D ) HEK293T cells expressing α1β2γ2 GABA A receptors were transfected with empty vector (EV) control or SERPINH1 cDNA plasmids ( C ), or transfected with non-targeting (NT) control siRNA or siRNA against SERPINH1 ( D ). Forty-eight hours post transfection, cycloheximide (CHX), a potent protein synthesis inhibitor, was added to the cell culture media for the indicated time. The remaining α1 protein levels were monitored and plotted against the CHX application time. Three biological replicates were performed. ( E ) HEK293T cells expressing α1β2γ2 GABA A receptors were transfected with non-targeting (NT) control siRNA or siRNA against SERPINH1 . Forty-eight hours post transfection, cells were lysed and the total proteins were immunoprecipitated with anti-α1 antibody. The eluents were probed with indicated antibodies. Three biological replicates were performed. Each data point is reported as mean ± SD. Statistical significance was calculated using two-tailed Student’s t-Test. *, p<0.05. Figure 3—figure supplement 1—source data 1. Original files for the western blot analysis in , C, D, and E. Figure 3—figure supplement 1—source data 2. PDF containing the original blots in with the relevant bands clearly labeled. Figure 3—figure supplement 1—source data 3. Data used for graphs presented in , B, and E.

Article Snippet: Antibody , anti-GABA A α1 (rabbit polyclonal) , Synaptic Systems , Cat#: 224203 , IF (1:250).

Techniques: Expressing, Transfection, Plasmid Preparation, Control, Immunoprecipitation, Cell Culture, Two Tailed Test, Western Blot, Labeling

Journal: eLife

Article Title: Hsp47 promotes biogenesis of multi-subunit neuroreceptors in the endoplasmic reticulum

doi: 10.7554/eLife.84798

Figure Lengend Snippet: HEK293T cells were transfected with α1, β2, and γ2 subunits of GABA A receptors, or α1-CFP, β2-YFP, and γ2 subunits. Forty-eight hours post-transfection, whole-cell patch-clamping electrophysiological recordings were carried out using IonFlux Mercury 16 ensemble plates to calculate EC 50 values for GABA (n=3–6 ensembles; each ensemble recording included 20 cells). The holding potential was set at –60 mV. Each data point is reported as mean ± SD.

Article Snippet: Antibody , anti-GABA A α1 (rabbit polyclonal) , Synaptic Systems , Cat#: 224203 , IF (1:250).

Techniques: Transfection

Journal: eLife

Article Title: Hsp47 promotes biogenesis of multi-subunit neuroreceptors in the endoplasmic reticulum

doi: 10.7554/eLife.84798

Figure Lengend Snippet: ( A ) Hsp47 overexpression increases FRET efficiency between CFP-tagged α1 subunit and YFP-tagged β2 subunit of GABA A receptors. HEK293T cells were transfected with CFP-tagged α1 subunit, YFP-tagged β2 subunit, and γ2 subunit; in addition, cells were transfected with empty vector (EV) control or Hsp47 cDNA. Forty-eight hours post transfection, pixel-based FRET was used to measure the FRET efficiency between α1-CFP and β2-YFP by using a confocal microscope. Representative images were shown for the CFP channel (1st columns), YFP channel (2nd columns), and FRET efficiency (3rd columns). Scale bar = 10 μm. Quantification of the FRET efficiency from 30 to 41 cells from at least three transfections was achieved using the ImageJ PixFRET plug-in, and shown on the right. ( B ) Overexpression of Hsp47 increases the interaction between α1 and β2 subunit of GABA A receptors. HEK293T cells stably expressing α1(Flag-β2)γ2 GABA A receptors were transfected with empty vector (EV) control or SERPINH1 cDNA. Forty-eight hours post transfection, Triton X-100 cell extracts were immunoprecipitated with a mouse anti-α1 antibody, and the immunoisolated eluents were subjected to immunoblotting assay. Three biological replicates were performed. Quantification of the relative intensity of Flag-β2 / α1 post IP is shown on the bottom. ( C ) HEK293T cells were transiently transfected with empty vector (EV), α1 subunits alone, or both α1 and β2 subunits of GABA A receptors together with SERPINH1 cDNA plasmids at various concentrations. Forty-eight hours post transfection, cells were lysed in RIPA buffer, and the total cell lysates were subjected to SDS-PAGE under non-reducing conditions and reducing conditions and immunoblotting analysis. Three biological replicates were performed. ( D ) Quantification of the 480 kDa band intensities for α1 and β2 subunits under non-reducing conditions (lanes 2–5 in C ) (n=3). ( E ) Quantification of the 50 kDa band intensities for α1 and β2 subunits under reducing conditions (lanes 7–10 in C ) (n=3). IP, immunoprecipitation; IB, immunoblotting. Each data point is reported as mean ± SD. Significant difference was analyzed by t-test ( A, B ) or a one-way ANOVA followed by post hoc Tukey’s HSD test ( D, E ). *, p<0.05; **, p<0.01; ***, p<0.001. Figure 4—source data 1. Original files for the western blot analysis in . Figure 4—source data 2. PDF containing the original blots in with the relevant bands clearly labeled. Figure 4—source data 3. Data used for graphs presented in .

Article Snippet: Antibody , anti-GABA A α1 (rabbit polyclonal) , Synaptic Systems , Cat#: 224203 , IF (1:250).

Techniques: Over Expression, Transfection, Plasmid Preparation, Control, Microscopy, Stable Transfection, Expressing, Immunoprecipitation, Western Blot, SDS Page, Labeling

Journal: eLife

Article Title: Hsp47 promotes biogenesis of multi-subunit neuroreceptors in the endoplasmic reticulum

doi: 10.7554/eLife.84798

Figure Lengend Snippet: ( A ) Overexpression of Hsp47 increases the endo H-resistant post-ER glycoform of the α1 subunit in HEK293T cells expressing α1(A322D)β2γ2 GABA A receptors. PNGase F treatment serves as a control for unglycosylated α1 subunit (lane 5). Two endo H-resistant bands were detected for the α1 subunit, indicated by the bracket (lanes 2 and 4). Three biological replicates were performed. Quantification of the ratio of endo H-resistant / total α1 subunit bands, as a measure of the ER-to-Golgi trafficking efficiency, is shown on the bottom. ( B ) HEK293T cells expressing α1(A322D)β2γ2 GABA A receptors were transfected with HA-ubiquitin together with empty vector (EV) control or SERPINH1 cDNA plasmids. Forty-eight hours post transfection, cells were lysed and the total proteins were immunoprecipitated with anti-α1 antibody. The eluents were probed with indicated antibodies. Three biological replicates were performed. ( C ) HEK293T cells expressing α1(A322D)β2γ2 GABA A receptors were transfected with empty vector (EV) control or SERPINH1 cDNA plasmids. Forty-eight hours post transfection, the surface proteins were measured using a cell surface protein biotinylation assay. The Na + /K + ATPase serves as a loading control for biotinylated membrane proteins. Alternatively, cells were lysed, and the total cell lysates were subjected to reducing SDS-PAGE and immunoblotting analysis. β-actin serves as a total protein loading control. Three biological replicates were performed. Protein intensities were quantified using ImageJ and shown on the bottom. ( D ) Whole-cell patch clamping was performed to record GABA-induced currents. HEK293T cells were treated as in ( C ). The recording was carried out 48 hr post transfection. The holding potential was set at –60 mV. Representative traces were shown. Quantification of the peak currents ( I max ) from 17 to 20 cells from three transfections is shown on the right. pA: picoampere. ( E ) Positions of the four α1 variants are displayed as space-filling models in the 3D structure of α1β2γ2 GABA A receptors, built from 6X3S.pdb using PyMOL. ( F ) HEK293T cells expressing α1(S76R)β2γ2, α1(D219N)β2γ2, or α1(G251D)β2γ2 GABA A receptors were transfected with EV control or SERPINH1 cDNA plasmids. Forty-eight hours post transfection, the surface proteins were measured using a cell surface protein biotinylation assay. Three biological replicates were performed. ( G ) Whole-cell patch clamping was performed to record GABA-induced currents using the IonFlux Mercury 16 ensemble plates at a holding voltage of −60 mV. HEK293T cells were treated as in ( F ). The recording was carried out 48 hr post transfection. Application of GABA (100 μM, 3 s) is indicated by the horizontal bar above the current traces. Each ensemble recording enclosed 20 cells. Quantification of the peak currents (I max ) is shown on the bottom (n=6–12 ensembles). Each data point is reported as mean ± SD. Statistical significance was calculated using two-tailed Student’s t-Test. *, p<0.05; **, p<0.01; ***, p<0.001. Figure 5—source data 1. Original files for the western blot analysis in . Figure 5—source data 2. PDF containing the original blots in with the relevant bands clearly labeled. Figure 5—source data 3. Data used for graphs presented in .

Article Snippet: Antibody , anti-GABA A α1 (rabbit polyclonal) , Synaptic Systems , Cat#: 224203 , IF (1:250).

Techniques: Over Expression, Expressing, Control, Transfection, Plasmid Preparation, Immunoprecipitation, Cell Surface Biotinylation Assay, Membrane, SDS Page, Western Blot, Two Tailed Test, Labeling

Journal: eLife

Article Title: Hsp47 promotes biogenesis of multi-subunit neuroreceptors in the endoplasmic reticulum

doi: 10.7554/eLife.84798

Figure Lengend Snippet: ( A, B ) HEK293T cells expressing α1(A322D)β2γ2 GABA A receptors were transfected with empty vector (EV) control or SERPINH1 cDNA plasmids ( A ), or transfected with non-targeting (NT) control siRNA or siRNA against SERPINH1 ( B ). Forty-eight hours post transfection, cycloheximide (CHX), a potent protein synthesis inhibitor, was added to the cell culture media for the indicated time. The remaining α1 protein levels were monitored and plotted against the CHX application time. Three biological replicates were performed. ( C ) HEK293T cells expressing α1(A322D)β2γ2 GABA A receptors were transfected with non-targeting (NT) control siRNA or siRNA against SERPINH1 . Forty-eight hours post transfection, cells were lysed and the total proteins were immunoprecipitated with anti-α1 antibody. The eluents were probed with indicated antibodies. Six biological replicates were performed. Each data point is reported as mean ± SD. Statistical significance was calculated using two-tailed Student’s t-Test. ***, p<0.001. Figure 5—figure supplement 1—source data 1. Original files for the western blot analysis in . Figure 5—figure supplement 1—source data 2. PDF containing the original blots in with the relevant bands clearly labeled. Figure 5—figure supplement 1—source data 3. Data used for graphs presented in .

Article Snippet: Antibody , anti-GABA A α1 (rabbit polyclonal) , Synaptic Systems , Cat#: 224203 , IF (1:250).

Techniques: Expressing, Transfection, Plasmid Preparation, Control, Cell Culture, Immunoprecipitation, Two Tailed Test, Western Blot, Labeling

Journal: eLife

Article Title: Hsp47 promotes biogenesis of multi-subunit neuroreceptors in the endoplasmic reticulum

doi: 10.7554/eLife.84798

Figure Lengend Snippet: HEK293T cells expressing α1(S76R)β2γ2, α1(D219N)β2γ2, or α1(G251D)β2γ2 GABA A receptors were transfected with empty vector (EV) control or SERPINH1 cDNA plasmids. Forty-eight hours post transfection, cells were lysed, and the total cell lysates were subjected to SDS-PAGE and immunoblotted for Hsp47. β-actin serves as a total protein loading control. Three biological replicates were performed. Quantification of Hsp47 protein levels was shown on the right. Each data point is reported as mean ± SD. Statistical significance was calculated using two-tailed Student’s t-Test. * p<0.05. Figure 5—figure supplement 2—source data 1. Original files for the western blot analysis in . Figure 5—figure supplement 2—source data 2. PDF containing the original blots in with the relevant bands clearly labeled. Figure 5—figure supplement 2—source data 3. Data used for graphs presented in .

Article Snippet: Antibody , anti-GABA A α1 (rabbit polyclonal) , Synaptic Systems , Cat#: 224203 , IF (1:250).

Techniques: Expressing, Transfection, Plasmid Preparation, Control, SDS Page, Two Tailed Test, Western Blot, Labeling

Journal: eLife

Article Title: Hsp47 promotes biogenesis of multi-subunit neuroreceptors in the endoplasmic reticulum

doi: 10.7554/eLife.84798

Figure Lengend Snippet: ( A ) HEK293T cells expressing WT α1β2γ2 or α1(A322D)β2γ2 GABA A receptors were transiently transfected with non-targeting (NT) control siRNAs or siRNAs against SERPINH1 (#1 or #2). Forty-eight hours post-transfection, cells were lysed for SDS-PAGE and Western blot analysis. ( B ) HEK293T cells expressing WT α1β2γ2 or α1(A322D)β2γ2 GABA A receptors were transiently transfected with empty vector (EV) or SERPINH1 cDNA plasmids. Forty-eight hours post-transfection, cells were lysed for SDS-PAGE and western blot analysis. Thapsigargin (Tg) (0.5 μM, 16 hr), a pan-UPR activator, was used as a positive control to induce the UPR. ( C ) Cultured cortical neurons from E18 rats were transduced with SERPINH1 siRNA lentivirus or scrambled siRNA lentivirus at days in vitro (DIV) 10. Forty-eight hours post transduction, neurons were lysed for SDS-PAGE and western blot analysis. ( D ) HEK293T cells expressing α1(A322D)β2γ2 GABA A receptors were transiently transfected with empty vector (EV), ATF6-N cDNA, or XBP1s cDNA plasmids for 48 hr, or treated with DMSO vehicle control or ATF6 activators (AA147 (10 μM) or AA263 (10 μM)) for 24 hr. Afterwards, cells were lysed for SDS-PAGE and western blot analysis. Three biological replicates were performed. Each data point is reported as mean ± SD. Significant difference was analyzed by a one-way ANOVA followed by post hoc Tukey’s HSD test ( A, B, D ) or t-test ( C ). *, p<0.05; **, p<0.01; ***, p<0.001. Figure 6—source data 1. Original files for the western blot analysis in . Figure 6—source data 2. PDF containing the original blots in with the relevant bands clearly labeled. Figure 6—source data 3. Data used for graphs presented in .

Article Snippet: Antibody , anti-GABA A α1 (rabbit polyclonal) , Synaptic Systems , Cat#: 224203 , IF (1:250).

Techniques: Expressing, Transfection, Control, SDS Page, Western Blot, Plasmid Preparation, Positive Control, Cell Culture, Transduction, In Vitro, Labeling

Journal: eLife

Article Title: Hsp47 promotes biogenesis of multi-subunit neuroreceptors in the endoplasmic reticulum

doi: 10.7554/eLife.84798

Figure Lengend Snippet:

Article Snippet: Antibody , anti-GABA A α1 (rabbit polyclonal) , Synaptic Systems , Cat#: 224203 , IF (1:250).

Techniques: Transfection, Construct, Control, Recombinant, Plasmid Preparation, Clone Assay, Software

Journal: bioRxiv

Article Title: Pharmacological chaperones restore proteostasis of epilepsy-associated GABA A receptor variants

doi: 10.1101/2023.04.18.537383

Figure Lengend Snippet: a Pathogenic probability of 149 clinical variants of the GABA A receptor α1 subunit is predicted using Rhapsody and plotted against its primary protein sequence. A schematic of the mature α1 sequence (residue 28-456) showing the N-terminal domain (NTD) and the transmembrane domain (M1-M4) is displayed on the bottom. The variants are classified into three categories: mild (pathogenic probability < 0.30), moderate (0.30 ≤ pathogenic probability < 0.60), and severe (pathogenic probability ≥ 0.60). Eight variants are selected for further characterization and colored in green. b The spatial distribution of the positions of α1 variants is illustrated in cryo-EM structure of α1β2γ2 GABA A receptors (6X3S.pdb), rendered using PyMOL. The C β of the residues (C α in the case of glycine) are shown as spheres. Mild variants are colored in blue, moderate variants in yellow, and severe variants in red. Eight selected variants are labelled. c HEK293T cells were transfected with α1 (wild type or the indicated variants), β2, and γ2 at a ratio of 1:1:1. Forty-eight hours post transfection, cells were lysed with a lysis buffer containing 2 mM DDM. The total proteins were subjected to SDS-PAGE and Western blot analysis. β-actin serves as a total protein loading control. Quantification of the normalized α1 band intensity is shown on the bottom (n = 3). d Surface biotinylation assay was used to quantify the surface α1 protein level. Na + /K + -ATPase serves as a plasma membrane protein loading control. Quantification of the normalized surface α1 band intensity is shown on the bottom (n = 3). e Insoluble α1 fraction was generated from removing soluble α1 fraction as shown in c by extracting proteins with a lysis buffer containing 2 mM DDM; residual insoluble α1 was re-suspended with Laemmli sample buffer containing 2% SDS and subjected to Western blot analysis. The band intensity was quantified by including the entire lane as indicated within the two arrows. Quantification of the ratio of insoluble over soluble α1 as a measure of aggregation propensity is shown on the bottom (n = 3). f Non-reducing protein gel was used to determine the oligomerization of α1 subunits. Total proteins were extracted and resolved through non-reducing SDS-PAGE in the absence of a reducing reagent. Quantification of oligomeric α1, as indicated within the two arrows, is shown on the bottom (n = 3). Each data point is reported as mean ± SEM. One-way ANOVA followed by post-hoc Tukey test was used for statistical analysis. *, p < 0.05; **, p < 0.01. Also see Supplementary Table S1 and Supplementary Fig. S2 .

Article Snippet: The mouse monoclonal anti-GABA A α1 subunit antibody (catalog #: 224211), rabbit polyclonal anti-GABA A α1 subunit antibody (catalog #: 224203), and rabbit polyclonal anti-GABA A γ2 antibody (catalog #: 224003) were obtained from Synaptic systems.

Techniques: Sequencing, Cryo-EM Sample Prep, Transfection, Lysis, SDS Page, Western Blot, Surface Biotinylation Assay, Generated

Journal: bioRxiv

Article Title: Pharmacological chaperones restore proteostasis of epilepsy-associated GABA A receptor variants

doi: 10.1101/2023.04.18.537383

Figure Lengend Snippet: a HEK293T cells were transiently transfected with α1 (wild type or the indicated variants), β2, and γ2 cDNAs of GABA A receptors. Forty-eight hours post transfection, whole-cell currents were recorded using the IonFlux Mercury 16 ensemble plates at a holding voltage of −60 mV. Representative whole-cell voltage-clamp recording traces are shown. Application of GABA (100 μM, 3 s) is indicated by the horizontal bar above the current traces. Quantification of the peak currents (I max ) is shown on the right (n = 4 to 10). nA: nano Ampere. b Dose-response curves of GABA are shown for the calculation of EC 50 values for wild type and the indicated α1 DAVs (n = 3 to 4). Each data point is reported as mean ± SEM. One-way ANOVA followed by post-hoc Tukey test was used for statistical analysis. ***, p < 0.001.

Article Snippet: The mouse monoclonal anti-GABA A α1 subunit antibody (catalog #: 224211), rabbit polyclonal anti-GABA A α1 subunit antibody (catalog #: 224203), and rabbit polyclonal anti-GABA A γ2 antibody (catalog #: 224003) were obtained from Synaptic systems.

Techniques: Transfection

Journal: bioRxiv

Article Title: Pharmacological chaperones restore proteostasis of epilepsy-associated GABA A receptor variants

doi: 10.1101/2023.04.18.537383

Figure Lengend Snippet: a-c Effect of Hispidulin (10 µM, 24 h) or TP003 (5 µM, 24 h) on the surface protein expression of the α1 variants in HEK293T cells stably expressing α1(D219N)β2γ2 ( a ), α1(G251D)β2γ2 ( b ), or α1(P260L)β2γ2 GABA A receptors ( c ) according to surface biotinylation analysis. Na + /K + ATPase serves as a plasma membrane protein loading control. Quantification of the surface α1 band intensities was shown on the bottom panels ( n = 3). d-f Effect of Hispidulin (10 µM, 24 h) or TP003 (5 µM, 24 h) on GABA-induced peak current amplitudes in HEK293T cells stably expressing α1(D219N)β2γ2 ( d ), α1(G251D)β2γ2 ( e ), or α1(P260L)β2γ2 GABA A receptors ( f ). Representative whole-cell voltage-clamp recording traces are shown. The holding voltage was set at −60 mV. Application of GABA (100 μM, 3 s) is indicated by the horizontal bar above the current traces. Quantification of the peak currents (I max ) are shown on the bottom panels (n = 4 to 8). pA: pico Ampere; nA: nano Ampere. g-i Dose-response curves of GABA are shown for the evaluation of the effect of Hispidulin (10 µM, 24 h) or TP003 (5 µM, 24 h) on α1(D219N)β2γ2 ( g ), α1(G251D)β2γ2 ( h ), or α1(P260L)β2γ2 GABA A receptors ( i ) (n = 3 to 4). Each data point is reported as mean ± SEM. One-way ANOVA followed by post-hoc Tukey test was used for statistical analysis. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The mouse monoclonal anti-GABA A α1 subunit antibody (catalog #: 224211), rabbit polyclonal anti-GABA A α1 subunit antibody (catalog #: 224203), and rabbit polyclonal anti-GABA A γ2 antibody (catalog #: 224003) were obtained from Synaptic systems.

Techniques: Expressing, Stable Transfection

Journal: bioRxiv

Article Title: Pharmacological chaperones restore proteostasis of epilepsy-associated GABA A receptor variants

doi: 10.1101/2023.04.18.537383

Figure Lengend Snippet: a,b Hispidulin (10 µM, 24 h) or TP003 (5 µM, 24 h) increases the FRET efficiency between CFP-tagged α1(G251D) subunit and YFP-tagged β2 subunit of GABA A receptors. HEK293T cells were transfected with CFP-tagged α1(G251D) subunit, YFP-tagged β2 subunit, and γ2 subunit; Twenty-four hours post transfection, cells were treated with DMSO, Hispidulin (10 µM) or TP003 (5 µM). Forty-eight hours post transfection, pixel-based FRET was used to calculate the FRET efficiency between α1(G251D)-CFP and β2-YFP. Representative images were shown for the CFP channel (1st columns), YFP channel (2nd columns), and FRET efficiency (3rd columns) ( a ). Scale bar = 20 μm. FRET efficiency was calculated from 30-40 cells from at least three transfections using the ImageJ PixFRET plug-in, shown in b . c Hispidulin (10 µM, 24 h) or TP003 (5 µM, 24 h) treatment increases the ER-to-Golgi trafficking efficiency of α1(G251D) in HEK293T cells stably expressing α1(G251D)β2γ2 GABA A receptors. The Peptide-N-Glycosidase F (PNGase F) enzyme cleavage serves as a control for unglycosylated α1 subunits (lane 7). After endo H digestion, α1 subunits with a molecular weight that is equal to unglycosylated α1 were labeled as endo H-sensitive, whereas those with higher molecular weights were labeled as endo H-resistant. Quantification of the ratio of endo H-resistant α1 / total α1 subunit bands, as a measure of the ER-to-Golgi trafficking efficiency, is shown on the bottom (n=3). d Effect of Hispidulin (10 µM, 24 h) and TP003 (5 µM, 24 h) on the protein levels of selected chaperones and γ2 subunits in HEK293T cells stably expressing α1(G251D)β2γ2 receptors (n=3). e Effect of Hispidulin (10 µM, 24 h) and TP003 (5 µM, 24 h) on the interactions between α1(G251D) and selected chaperones or γ2 in HEK293T cells stably expressing α1(G251D)β2γ2 receptors. Quantification of the ratio of proteins of interest / α1(G251D) post immunoprecipitation is shown in the right panels (n=3). IP: immunoprecipitation. f Effect of Hispidulin (10 µM, 24 h) and TP003 (5 µM, 24 h) on the mRNA levels of genes of interest in HEK293T cells stably expressing α1(G251D)β2γ2 receptors, assessed by quantitative RT-PCR (n=3). Each data point is reported as mean ± SEM. One-way ANOVA followed by post-hoc Tukey test was used for statistical analysis. * p < 0.05; ** p < 0.01; *** p < 0.001. Also see Supplementary Table S2 .

Article Snippet: The mouse monoclonal anti-GABA A α1 subunit antibody (catalog #: 224211), rabbit polyclonal anti-GABA A α1 subunit antibody (catalog #: 224203), and rabbit polyclonal anti-GABA A γ2 antibody (catalog #: 224003) were obtained from Synaptic systems.

Techniques: Transfection, Stable Transfection, Expressing, Molecular Weight, Labeling, Immunoprecipitation, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Pharmacological chaperones restore proteostasis of epilepsy-associated GABA A receptor variants

doi: 10.1101/2023.04.18.537383

Figure Lengend Snippet: a Schematic of the generation of α1(G215D) knockin in hiPSCs and the transcription factor (TF)-based differentiation into GABAergic neurons. b Effect of Hispidulin (10 µM, 24 h) and TP003 (5 µM, 24 h) on the surface expression of GABA A receptor subunits in hiPSC-derived GABAergic neurons carrying the α1(G215D) variant. Surface GABA A receptors were stained using anti-α1 subunit, anti-β2/β3 subunit, or anti-γ2 subunit antibodies without membrane permeabilization. 50-75 neurons from at least three differentiations were imaged by confocal microscopy for each condition. Scale bar = 20 μm. c Quantification of the fluorescence intensity of the surface GABA A receptor subunits after background correction. d Effect of Hispidulin (10 µM, 24 h) and TP003 (5 µM, 24 h) on the interactions between α1(G251D) and selected ER proteostasis network components. PLA was used to determine the endogenous protein-protein interactions. 35-70 neurons from at least three differentiations were imaged by confocal microscopy for each condition. Scale bar = 20 μm. e Quantification of the PLA puncta number per cell was achieved using the ImageJ Analyze Particles plug-in. Each data point is reported as mean ± SEM. ANOVA followed by post-hoc Tukey test was used to evaluate the statistical significance. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Also see Supplementary Fig. S5 .

Article Snippet: The mouse monoclonal anti-GABA A α1 subunit antibody (catalog #: 224211), rabbit polyclonal anti-GABA A α1 subunit antibody (catalog #: 224203), and rabbit polyclonal anti-GABA A γ2 antibody (catalog #: 224003) were obtained from Synaptic systems.

Techniques: Knock-In, Expressing, Derivative Assay, Variant Assay, Staining, Confocal Microscopy, Fluorescence