mouse monoclonal anti hsp47  (Novus Biologicals)

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: The mouse monoclonal anti-Hsp47 antibody (catalog #: ADI-SPA-470-F) came from Enzo Life Sciences (Farmingdale, NY).

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: The mouse monoclonal anti-Hsp47 antibody (catalog #: ADI-SPA-470-F) came from Enzo Life Sciences (Farmingdale, NY).

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: The mouse monoclonal anti-Hsp47 antibody (catalog #: ADI-SPA-470-F) came from Enzo Life Sciences (Farmingdale, NY).

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: The mouse monoclonal anti-Hsp47 antibody (catalog #: ADI-SPA-470-F) came from Enzo Life Sciences (Farmingdale, NY).

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: The mouse monoclonal anti-Hsp47 antibody (catalog #: ADI-SPA-470-F) came from Enzo Life Sciences (Farmingdale, NY).

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 ) 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: The mouse monoclonal anti-Hsp47 antibody (catalog #: ADI-SPA-470-F) came from Enzo Life Sciences (Farmingdale, NY).

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: The mouse monoclonal anti-Hsp47 antibody (catalog #: ADI-SPA-470-F) came from Enzo Life Sciences (Farmingdale, NY).

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: 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: The mouse monoclonal anti-Hsp47 antibody (catalog #: ADI-SPA-470-F) came from Enzo Life Sciences (Farmingdale, NY).

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 were transfected with nAChR subunits (α4 ( CHRNA4 ) and β2 ( CHRNB2 )) and empty vector (EV) control or SERPINH1 cDNA plasmids. Forty-eight hours post transfection, cells were lysed and the total proteins were evaluated using a western blot analysis. ( B ) Hsp47 overexpression increases FRET efficiency between CFP-tagged β2 subunit and YFP-tagged α4 subunit of nAChRs. HEK293T cells were transfected with CFP-tagged β2 subunit and YFP-tagged α4 subunit; in addition, cells were transfected with empty vector (EV) control or SERPINH1 cDNA. Forty-eight hours post transfection, pixel-based FRET was used to measure the FRET efficiency between β2-CFP and α4-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 60 to 70 cells from at least three transfections was achieved using the ImageJ PixFRET plug-in, and shown on the right. ( C ) HEK293T cells were transfected with CFP-tagged β2 subunit and YFP-tagged α4 subunit of nAChRs; in addition, cells were transfected with empty vector (EV) control or SERPINH1 cDNA. Forty-eight hours post transfection, whole-cell patch clamping was performed to record nicotine-induced currents. Representative traces were shown. Quantification of the peak currents ( I max ) from 9 cells from three transfections is shown on the right. The holding potential was set at –60 mV. pA: picoampere. ( D ) HEK293T cells were transfected with 5-HT 3 R subunits (5-HT 3 A and FLAG-tagged 5-HT 3 B) and 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, and the total proteins were evaluated using a Western blot analysis. The Na + /K + ATPase serves as a loading control for biotinylated membrane proteins. 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; ** p<0.01; *** p<0.001. Figure 7—source data 1. Original files for the western blot analysis in . Figure 7—source data 2. PDF containing the original blots in with the relevant bands clearly labeled. Figure 7—source data 3. Data used for graphs presented in .

Article Snippet: The mouse monoclonal anti-Hsp47 antibody (catalog #: ADI-SPA-470-F) came from Enzo Life Sciences (Farmingdale, NY).

Techniques: Transfection, Plasmid Preparation, Control, Western Blot, Over Expression, Microscopy, Cell Surface Biotinylation Assay, Membrane, 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 ) Hsp47 overexpression does not change the FRET efficiency between Cerulean (an improved CFP variant)-tagged α7 subunit and Venus (an improved YFP variant)-tagged α7 subunit of nAChRs. HEK293T cells were transfected with Cerulean-tagged α7 subunit and Venus-tagged α7 subunit at a 1:1 ratio; in addition, cells were transfected with empty vector (EV) control or SERPINH1 cDNA. Forty-eight hours post transfection, pixel-based FRET was used to measure the FRET efficiency between α7-Cerulean and α7-Venus 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 90 to 105 cells from at least three transfections was achieved using the ImageJ PixFRET plug-in, and shown on the right. ( B ) HEK293T cells were transfected with nAChR α7 subunits and empty vector (EV) control or SERPINH1 cDNA plasmids. Forty-eight hours post transfection, cells were lysed and the total proteins were evaluated using a western blot analysis. Three biological replicates were performed. Each data point is reported as mean ± SD. Statistical significance was calculated using two-tailed Student’s t-Test. NS, not significant; ** p<0.01. Figure 7—figure supplement 1—source data 1. Original files for the Western blot analysis in . Figure 7—figure supplement 1—source data 2. PDF containing the original blots in with the relevant bands clearly labeled. Figure 7—figure supplement 1—source data 3. Data used for graphs presented in .

Article Snippet: The mouse monoclonal anti-Hsp47 antibody (catalog #: ADI-SPA-470-F) came from Enzo Life Sciences (Farmingdale, NY).

Techniques: Over Expression, Variant Assay, Transfection, Plasmid Preparation, Control, Microscopy, 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: BiP and calnexin assist the subunit folding early in the ER lumen. Hsp47 operates after BiP and binds the folded states of the α1 or β subunits in the ER lumen. Hsp47 links the α1 and β subunits and promotes their inter-subunit interactions. As a result, Hsp47 promotes the formation of assembly intermediates and the native pentameric receptors in the ER. Assembled receptors will traffic to the Golgi and onward to the plasma membrane for function.

Article Snippet: The mouse monoclonal anti-Hsp47 antibody (catalog #: ADI-SPA-470-F) came from Enzo Life Sciences (Farmingdale, NY).

Techniques: Membrane

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 mouse monoclonal anti-Hsp47 antibody (catalog #: ADI-SPA-470-F) came from Enzo Life Sciences (Farmingdale, NY).

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: ( 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: The rabbit polyclonal anti-nAChR β2 subunit ( CHRNB2 ) antibody (catalog #: 17844–1-AP), rabbit polyclonal anti-nAChR α7 subunit ( CHRNA7 ) antibody (catalog #: 21379–1-AP), rabbit polyclonal anti-ATF6 antibody (catalog # 24169–1-AP), rabbit polyclonal anti-FLAG antibody (catalog #: 20543–1-AP), and mouse monoclonal anti-Hsp47 antibody (catalog #: 67863–1-lg) were purchased from Proteintech (Rosemont, IL, USA).

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: The rabbit polyclonal anti-nAChR β2 subunit ( CHRNB2 ) antibody (catalog #: 17844–1-AP), rabbit polyclonal anti-nAChR α7 subunit ( CHRNA7 ) antibody (catalog #: 21379–1-AP), rabbit polyclonal anti-ATF6 antibody (catalog # 24169–1-AP), rabbit polyclonal anti-FLAG antibody (catalog #: 20543–1-AP), and mouse monoclonal anti-Hsp47 antibody (catalog #: 67863–1-lg) were purchased from Proteintech (Rosemont, IL, USA).

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: The rabbit polyclonal anti-nAChR β2 subunit ( CHRNB2 ) antibody (catalog #: 17844–1-AP), rabbit polyclonal anti-nAChR α7 subunit ( CHRNA7 ) antibody (catalog #: 21379–1-AP), rabbit polyclonal anti-ATF6 antibody (catalog # 24169–1-AP), rabbit polyclonal anti-FLAG antibody (catalog #: 20543–1-AP), and mouse monoclonal anti-Hsp47 antibody (catalog #: 67863–1-lg) were purchased from Proteintech (Rosemont, IL, USA).

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: The rabbit polyclonal anti-nAChR β2 subunit ( CHRNB2 ) antibody (catalog #: 17844–1-AP), rabbit polyclonal anti-nAChR α7 subunit ( CHRNA7 ) antibody (catalog #: 21379–1-AP), rabbit polyclonal anti-ATF6 antibody (catalog # 24169–1-AP), rabbit polyclonal anti-FLAG antibody (catalog #: 20543–1-AP), and mouse monoclonal anti-Hsp47 antibody (catalog #: 67863–1-lg) were purchased from Proteintech (Rosemont, IL, USA).

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: The rabbit polyclonal anti-nAChR β2 subunit ( CHRNB2 ) antibody (catalog #: 17844–1-AP), rabbit polyclonal anti-nAChR α7 subunit ( CHRNA7 ) antibody (catalog #: 21379–1-AP), rabbit polyclonal anti-ATF6 antibody (catalog # 24169–1-AP), rabbit polyclonal anti-FLAG antibody (catalog #: 20543–1-AP), and mouse monoclonal anti-Hsp47 antibody (catalog #: 67863–1-lg) were purchased from Proteintech (Rosemont, IL, USA).

Techniques: Over Expression, Stable Transfection, Expressing, Control, Glycoproteomics, 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 ) 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: The rabbit polyclonal anti-nAChR β2 subunit ( CHRNB2 ) antibody (catalog #: 17844–1-AP), rabbit polyclonal anti-nAChR α7 subunit ( CHRNA7 ) antibody (catalog #: 21379–1-AP), rabbit polyclonal anti-ATF6 antibody (catalog # 24169–1-AP), rabbit polyclonal anti-FLAG antibody (catalog #: 20543–1-AP), and mouse monoclonal anti-Hsp47 antibody (catalog #: 67863–1-lg) were purchased from Proteintech (Rosemont, IL, USA).

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: The rabbit polyclonal anti-nAChR β2 subunit ( CHRNB2 ) antibody (catalog #: 17844–1-AP), rabbit polyclonal anti-nAChR α7 subunit ( CHRNA7 ) antibody (catalog #: 21379–1-AP), rabbit polyclonal anti-ATF6 antibody (catalog # 24169–1-AP), rabbit polyclonal anti-FLAG antibody (catalog #: 20543–1-AP), and mouse monoclonal anti-Hsp47 antibody (catalog #: 67863–1-lg) were purchased from Proteintech (Rosemont, IL, USA).

Techniques: Over Expression, Expressing, Control, Transfection, Ubiquitin Proteomics, 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: 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: The rabbit polyclonal anti-nAChR β2 subunit ( CHRNB2 ) antibody (catalog #: 17844–1-AP), rabbit polyclonal anti-nAChR α7 subunit ( CHRNA7 ) antibody (catalog #: 21379–1-AP), rabbit polyclonal anti-ATF6 antibody (catalog # 24169–1-AP), rabbit polyclonal anti-FLAG antibody (catalog #: 20543–1-AP), and mouse monoclonal anti-Hsp47 antibody (catalog #: 67863–1-lg) were purchased from Proteintech (Rosemont, IL, USA).

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 were transfected with nAChR subunits (α4 ( CHRNA4 ) and β2 ( CHRNB2 )) and empty vector (EV) control or SERPINH1 cDNA plasmids. Forty-eight hours post transfection, cells were lysed and the total proteins were evaluated using a western blot analysis. ( B ) Hsp47 overexpression increases FRET efficiency between CFP-tagged β2 subunit and YFP-tagged α4 subunit of nAChRs. HEK293T cells were transfected with CFP-tagged β2 subunit and YFP-tagged α4 subunit; in addition, cells were transfected with empty vector (EV) control or SERPINH1 cDNA. Forty-eight hours post transfection, pixel-based FRET was used to measure the FRET efficiency between β2-CFP and α4-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 60 to 70 cells from at least three transfections was achieved using the ImageJ PixFRET plug-in, and shown on the right. ( C ) HEK293T cells were transfected with CFP-tagged β2 subunit and YFP-tagged α4 subunit of nAChRs; in addition, cells were transfected with empty vector (EV) control or SERPINH1 cDNA. Forty-eight hours post transfection, whole-cell patch clamping was performed to record nicotine-induced currents. Representative traces were shown. Quantification of the peak currents ( I max ) from 9 cells from three transfections is shown on the right. The holding potential was set at –60 mV. pA: picoampere. ( D ) HEK293T cells were transfected with 5-HT 3 R subunits (5-HT 3 A and FLAG-tagged 5-HT 3 B) and 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, and the total proteins were evaluated using a Western blot analysis. The Na + /K + ATPase serves as a loading control for biotinylated membrane proteins. 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; ** p<0.01; *** p<0.001. Figure 7—source data 1. Original files for the western blot analysis in . Figure 7—source data 2. PDF containing the original blots in with the relevant bands clearly labeled. Figure 7—source data 3. Data used for graphs presented in .

Article Snippet: The rabbit polyclonal anti-nAChR β2 subunit ( CHRNB2 ) antibody (catalog #: 17844–1-AP), rabbit polyclonal anti-nAChR α7 subunit ( CHRNA7 ) antibody (catalog #: 21379–1-AP), rabbit polyclonal anti-ATF6 antibody (catalog # 24169–1-AP), rabbit polyclonal anti-FLAG antibody (catalog #: 20543–1-AP), and mouse monoclonal anti-Hsp47 antibody (catalog #: 67863–1-lg) were purchased from Proteintech (Rosemont, IL, USA).

Techniques: Transfection, Plasmid Preparation, Control, Western Blot, Over Expression, Microscopy, Cell Surface Biotinylation Assay, Membrane, 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 ) Hsp47 overexpression does not change the FRET efficiency between Cerulean (an improved CFP variant)-tagged α7 subunit and Venus (an improved YFP variant)-tagged α7 subunit of nAChRs. HEK293T cells were transfected with Cerulean-tagged α7 subunit and Venus-tagged α7 subunit at a 1:1 ratio; in addition, cells were transfected with empty vector (EV) control or SERPINH1 cDNA. Forty-eight hours post transfection, pixel-based FRET was used to measure the FRET efficiency between α7-Cerulean and α7-Venus 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 90 to 105 cells from at least three transfections was achieved using the ImageJ PixFRET plug-in, and shown on the right. ( B ) HEK293T cells were transfected with nAChR α7 subunits and empty vector (EV) control or SERPINH1 cDNA plasmids. Forty-eight hours post transfection, cells were lysed and the total proteins were evaluated using a western blot analysis. Three biological replicates were performed. Each data point is reported as mean ± SD. Statistical significance was calculated using two-tailed Student’s t-Test. NS, not significant; ** p<0.01. Figure 7—figure supplement 1—source data 1. Original files for the Western blot analysis in . Figure 7—figure supplement 1—source data 2. PDF containing the original blots in with the relevant bands clearly labeled. Figure 7—figure supplement 1—source data 3. Data used for graphs presented in .

Article Snippet: The rabbit polyclonal anti-nAChR β2 subunit ( CHRNB2 ) antibody (catalog #: 17844–1-AP), rabbit polyclonal anti-nAChR α7 subunit ( CHRNA7 ) antibody (catalog #: 21379–1-AP), rabbit polyclonal anti-ATF6 antibody (catalog # 24169–1-AP), rabbit polyclonal anti-FLAG antibody (catalog #: 20543–1-AP), and mouse monoclonal anti-Hsp47 antibody (catalog #: 67863–1-lg) were purchased from Proteintech (Rosemont, IL, USA).

Techniques: Over Expression, Variant Assay, Transfection, Plasmid Preparation, Control, Microscopy, 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: BiP and calnexin assist the subunit folding early in the ER lumen. Hsp47 operates after BiP and binds the folded states of the α1 or β subunits in the ER lumen. Hsp47 links the α1 and β subunits and promotes their inter-subunit interactions. As a result, Hsp47 promotes the formation of assembly intermediates and the native pentameric receptors in the ER. Assembled receptors will traffic to the Golgi and onward to the plasma membrane for function.

Article Snippet: The rabbit polyclonal anti-nAChR β2 subunit ( CHRNB2 ) antibody (catalog #: 17844–1-AP), rabbit polyclonal anti-nAChR α7 subunit ( CHRNA7 ) antibody (catalog #: 21379–1-AP), rabbit polyclonal anti-ATF6 antibody (catalog # 24169–1-AP), rabbit polyclonal anti-FLAG antibody (catalog #: 20543–1-AP), and mouse monoclonal anti-Hsp47 antibody (catalog #: 67863–1-lg) were purchased from Proteintech (Rosemont, IL, USA).

Techniques: Clinical Proteomics, Membrane

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-nAChR β2 subunit ( CHRNB2 ) antibody (catalog #: 17844–1-AP), rabbit polyclonal anti-nAChR α7 subunit ( CHRNA7 ) antibody (catalog #: 21379–1-AP), rabbit polyclonal anti-ATF6 antibody (catalog # 24169–1-AP), rabbit polyclonal anti-FLAG antibody (catalog #: 20543–1-AP), and mouse monoclonal anti-Hsp47 antibody (catalog #: 67863–1-lg) were purchased from Proteintech (Rosemont, IL, USA).

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

Journal: bioRxiv

Article Title: Hsp47 Promotes Biogenesis of Multi-subunit Neuroreceptors in the Endoplasmic Reticulum

doi: 10.1101/2022.10.24.513629

Figure Lengend Snippet: Hsp47 interacts with GABA A receptors. ( A ) Endogenous interactions between GABA A receptor α1 subunits and Hsp47. Mouse brain homogenates from 8-10 weeks C57BL/6J mice were immunoprecipitated with an anti-α1 antibody, and the immunoisolated eluents were blotted with indicated antibodies (n = 3). IgG was included as a negative control for non-specific binding. ( 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 (n = 3). ( C ) MicroScale Thermophoresis (MST) was used to determine the binding affinities between ER luminal chaperones (Hsp47 and BiP) to RED-labeled His-α1(ERD). Increasing concentrations of recombinant Hsp47 or BiP proteins (0.20 nM – 10 μM) were incubated with 50 nM RED-labeled His-α1(ERD) protein in PBS with Tween-20 (0.05%) (n = 3). 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. The data were analyzed using the Monolith software for the calculation of the dissociation constant (Kd). IP, immunoprecipitation; IB, immunoblotting.

Article Snippet: The mouse monoclonal anti-Hsp47 antibody (catalog #: ADI-SPA-470-F) came from Enzo Life Sciences (Farmingdale, NY).

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

Journal: bioRxiv

Article Title: Hsp47 Promotes Biogenesis of Multi-subunit Neuroreceptors in the Endoplasmic Reticulum

doi: 10.1101/2022.10.24.513629

Figure Lengend Snippet: Hsp47 positively regulates the surface expression of endogenous GABA A receptors in cultured neurons. ( A ) Effect of Hsp47 on the surface expression of endogenous GABA A receptor subunits in primary rat hippocampal neurons. Cultured neurons were transduced with Hsp47 siRNA lentivirus or scrambled siRNA lentivirus at days in vitro (DIV) 10. Forty-eight hours post transduction, surface GABA A receptors were stained using anti-α1 subunit or anti-β2/β3 subunit antibodies without membrane permeabilization. The cells were then washed, and permeabilized before we stained the nuclei with DAPI. 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 of panel A. Scale bar = 10 μm. Quantification of the fluorescence intensity of the surface GABA A receptor subunits after background correction was shown on the right. ( B ) Whole-cell patch clamping was performed to record GABA-induced currents. Neurons were subjected to transduction as in ( A ). The recordings were carried out 48 hours post transduction. Representative traces are shown in the left-hand panel. Peak current amplitude ( I max ) is shown on the right (n = 10). The holding potential was set at −60 mV. pA: picoampere. Each data point is reported as mean ± SD. Statistical significance was calculated using an unpaired two-tailed Student’s t-Test. *** p < 0.001.

Article Snippet: The mouse monoclonal anti-Hsp47 antibody (catalog #: ADI-SPA-470-F) came from Enzo Life Sciences (Farmingdale, NY).

Techniques: Expressing, Cell Culture, Transduction, In Vitro, Staining, Confocal Microscopy, Fluorescence, Two Tailed Test

Journal: bioRxiv

Article Title: Hsp47 Promotes Biogenesis of Multi-subunit Neuroreceptors in the Endoplasmic Reticulum

doi: 10.1101/2022.10.24.513629

Figure Lengend Snippet: Hsp47 preferentially binds the folded conformation of GABA A receptor subunits. ( 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. 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). 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 (n = 3). ( 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 minutes. Then Triton X-100 cell extracts were immunoprecipitated with a mouse anti-Hsp47 antibody, and the immunoisolated eluents were subjected for immunoblotting assay (n = 3). 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 (n = 3). 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 (n = 3). 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 (n = 3). 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 (n = 3). Quantification of the relative intensity of BiP/α1 post IP is shown on the bottom panel. IP, immunoprecipitation; IB, immunoblotting. 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.

Article Snippet: The mouse monoclonal anti-Hsp47 antibody (catalog #: ADI-SPA-470-F) came from Enzo Life Sciences (Farmingdale, NY).

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

Journal: bioRxiv

Article Title: Hsp47 Promotes Biogenesis of Multi-subunit Neuroreceptors in the Endoplasmic Reticulum

doi: 10.1101/2022.10.24.513629

Figure Lengend Snippet: Hsp47 promotes the assembly of GABA A receptors. ( 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 30-41 cells 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 Hsp47 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. Quantification of the relative intensity of Flag-β2 / α1 post IP is shown on the bottom (n = 3). ( 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 Hsp47 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. ( D ) Quantification of the 480 kDa band intensities for α1 and β2 subunits under nonreducing 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.

Article Snippet: The mouse monoclonal anti-Hsp47 antibody (catalog #: ADI-SPA-470-F) came from Enzo Life Sciences (Farmingdale, NY).

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

Journal: bioRxiv

Article Title: Hsp47 Promotes Biogenesis of Multi-subunit Neuroreceptors in the Endoplasmic Reticulum

doi: 10.1101/2022.10.24.513629

Figure Lengend Snippet: Hsp47 positively regulates the functional surface expression of epilepsy-associated GABA A receptors carrying the α1(A322D) variant. ( 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). 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 (n = 3). ( B ) HEK293T cells expressing α1(A322D)β2γ2 GABA A receptors were transfected with empty vector (EV) control or Hsp47 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. Surface α1 subunit intensities were quantified using ImageJ and shown on the bottom ( n = 3). Alternatively, 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. ( C ) Whole-cell patch clamping was performed to record GABA-induced currents. HEK293T cells were treated as in ( B ). The recording was carried out 48 hours post transfection. The holding potential was set at −60 mV. Representative traces were shown. Quantification of the peak currents ( I max ) from 17-20 cells is shown on the bottom. pA: picoampere. 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.

Article Snippet: The mouse monoclonal anti-Hsp47 antibody (catalog #: ADI-SPA-470-F) came from Enzo Life Sciences (Farmingdale, NY).

Techniques: Functional Assay, Expressing, Variant Assay, Over Expression, Transfection, Plasmid Preparation, Cell Surface Biotinylation Assay, SDS Page, Two Tailed Test

Journal: bioRxiv

Article Title: Hsp47 Promotes Biogenesis of Multi-subunit Neuroreceptors in the Endoplasmic Reticulum

doi: 10.1101/2022.10.24.513629

Figure Lengend Snippet: Hsp47 promotes the assembly and function of α4β2 nicotinic acetylcholine receptors (nAChRs). ( A ) Hsp47 overexpression increases FRET efficiency between CFP-tagged β2 subunit and YFP-tagged α4 subunit of nAChRs. HEK293T cells were transfected with CFP-tagged β2 subunit and YFP-tagged α4 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 β2-CFP and α4-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 60-70 cells was achieved using the ImageJ PixFRET plug-in, and shown on the right. ( B ) HEK293T cells were transfected with CFP-tagged β2 subunit and YFP-tagged α4 subunit of nAChRs; in addition, cells were transfected with empty vector (EV) control or Hsp47 cDNA. Forty-eight hours post transfection, whole-cell patch clamping was performed to record nicotine-induced currents. Representative traces were shown. Quantification of the peak currents ( I max ) from 9 cells 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 two-tailed Student’s t-Test. * p < 0.05; *** p < 0.001.

Article Snippet: The mouse monoclonal anti-Hsp47 antibody (catalog #: ADI-SPA-470-F) came from Enzo Life Sciences (Farmingdale, NY).

Techniques: Over Expression, Transfection, Plasmid Preparation, Microscopy, Two Tailed Test

Journal: bioRxiv

Article Title: Hsp47 Promotes Biogenesis of Multi-subunit Neuroreceptors in the Endoplasmic Reticulum

doi: 10.1101/2022.10.24.513629

Figure Lengend Snippet: Proposed mechanism of Hsp47 in the assembly of GABA A receptors. BiP and calnexin assist the subunit folding early in the ER lumen. Hsp47 operates after BiP and binds the folded states of the α1 or β subunits in the ER lumen. Hsp47 links the α1 and β subunits and promotes their inter-subunit interactions. As a result, Hsp47 promotes the formation of assembly intermediates and the native pentameric receptors in the ER. Assembled receptors will traffic to the Golgi and onward to the plasma membrane for function.

Article Snippet: The mouse monoclonal anti-Hsp47 antibody (catalog #: ADI-SPA-470-F) came from Enzo Life Sciences (Farmingdale, NY).

Techniques: