Journal: Journal of translational medicine

Article Title: ESRRG-PKM2 axis reprograms metabolism to suppress esophageal squamous carcinoma progression and enhance anti-PD-1 therapy efficacy.

doi: 10.1186/s12967-023-04347-5

Figure Lengend Snippet: Fig. 4 ESRRG inhibits the expression of key glycolytic enzyme PKM2 in ESCC cells. A A schematic diagram illustrates the regulation of glycolysis pathway. B Heatmap illustrates glycolysis related genes in transcript levels between TE1 cells with ESRRG overexpression and control. C The correlation between the relative levels of ESRRG and LDHA, PFKM, GPI, GAPDH, ENO1, PKM, TPI1, ALDOA mRNA transcripts in 82 ESCC tissues of TCGA database. D mRNA levels of 8 glycolysis-related genes in TE1 with ESRRG overexpression and ECa109 cells with ESRRG knockdown compared to relative control cells. E The correlation between the relative levels of ESRRG and PKM, determined by RT-PCR in 94 ESCC tissues. F Schematics of mutation strategies in the PKM promoter (− 2000 bp to + 50 bp). G Luciferase reporter assays exhibited that ESRRG bound to the PKM promoter at site 2 to induce its expression. H Effects of ESRRG overexpression and knockdown on PKM2 expression in ESCC cells. Immunohistochemistry (I) and Western blotting (J) for PKM2 protein level in xenograft tumor of mice after overexpression or knockout of ESRRG. Values are presented as mean ± SD (n = 3). *P < 0.05 or **P < 0.01 indicates significant differences from the vehicle group as assessed by a one-way ANOVA with a post hoc Dunnett’s test

Article Snippet: ESRRG (Proteintech, Cat No. 14017- 1-AP), PKM2 (Cell Signaling Technology, Cat No. 4053), β-actin (Cell Signaling Technology, Cat No. 4970), CD8 (Cell Signaling Technology, Cat No. 85336), and GAPDH (Santa Cruz Biotechnology, Cat No. sc47724) were used as primary antibodies.

Techniques: Expressing, Over Expression, Control, Knockdown, Reverse Transcription Polymerase Chain Reaction, Mutagenesis, Luciferase, Immunohistochemistry, Western Blot, Knock-Out

Journal: Journal of translational medicine

Article Title: ESRRG-PKM2 axis reprograms metabolism to suppress esophageal squamous carcinoma progression and enhance anti-PD-1 therapy efficacy.

doi: 10.1186/s12967-023-04347-5

Figure Lengend Snippet: Fig. 5 PKM2 is essential for ESRRG to inhibits tumor growth in ESCC cells. A–D Cell growth were determined in ESRRG knockdown ECa109 and KYSE510 cells with or without further knockdown of PKM employing CCK-8 assays(A, colony formation B and EDU assay (C, D). E Typical pictures of tumors isolated from nude mice with tumor xenografts derived from the indicated groups. F Tumour volumes were monitored at indicated time points. G The weight of tumours was measured at time of sacrificed. Values are presented as mean ± SD (n = 3–5). *P < 0.05 or **P < 0.01 indicates significant differences from the vehicle group as assessed by a one-way ANOVA with a post hoc Dunnett’s test

Article Snippet: ESRRG (Proteintech, Cat No. 14017- 1-AP), PKM2 (Cell Signaling Technology, Cat No. 4053), β-actin (Cell Signaling Technology, Cat No. 4970), CD8 (Cell Signaling Technology, Cat No. 85336), and GAPDH (Santa Cruz Biotechnology, Cat No. sc47724) were used as primary antibodies.

Techniques: Knockdown, CCK-8 Assay, EdU Assay, Isolation, Derivative Assay

Journal: Journal of translational medicine

Article Title: ESRRG-PKM2 axis reprograms metabolism to suppress esophageal squamous carcinoma progression and enhance anti-PD-1 therapy efficacy.

doi: 10.1186/s12967-023-04347-5

Figure Lengend Snippet: Fig. 8 Correlation of ESRRG and PKM2 in ESCC. A IHC analysis for ESRRG and PKM2 expression in clinical HCC samples. Scale bars = 100 μm. B Correlation between ESRRG expression and PKM2 expression (Chi-squared test). C Prognostic value of combining ESRRG and PKM2 levels was analyzed by Kaplan–Meier analysis in 94 ESCC samples. D Model illustrating tumor suppression mechanism regulated by ESRRG in ESCC progression

Article Snippet: ESRRG (Proteintech, Cat No. 14017- 1-AP), PKM2 (Cell Signaling Technology, Cat No. 4053), β-actin (Cell Signaling Technology, Cat No. 4970), CD8 (Cell Signaling Technology, Cat No. 85336), and GAPDH (Santa Cruz Biotechnology, Cat No. sc47724) were used as primary antibodies.

Techniques: Expressing

Journal: Frontiers in Neuroscience

Article Title: Long-term aerobic exercise improves learning memory capacity and effects on oxidative stress levels and Keap1/Nrf2/GPX4 pathway in the hippocampus of APP/PS1 mice

doi: 10.3389/fnins.2024.1505650

Figure Lengend Snippet: Aerobic exercise regulates Keap1/Nrf2/GPX4 pathway of the hippocampus in APP/AS1 mice. (A) Representative western blots of Keap1, Nrf2, HO-1, and GPX4 expression. (B–E) Quantification data of Keap1, Nrf2, HO-1, and GPX4 protein expression. n = 4, ∗ p < 0.05, compared with the control group; # p < 0.05, compared with the model group; & p < 0.05, compared with the S-te group. C, control group; M, model group; S-te, short-term exercise group; L-te, long-term exercise group.

Article Snippet: The protein sample was then electrophoresed in 12% SDS-polyacrylamide gels, transferred onto the PVDF membrane, and blocked using 5% skimmed milk for 1 h. The membrane was incubated with diluted (1:1000) primary antibodies against Keap-1 antibody (ab178846; Abcam), Nrf2 antibody 28,205-1-AP; Proteintech, HO-1 antibody (13129-1-AP; Proteintech, Shanghai, China), or GPX4 antibody (14104-1-AP; Proteintech, Shanghai, China) at 4°C overnight.

Techniques: Western Blot, Expressing, Control

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 pCMV6 plasmids containing human GABA A receptor α1 subunit ( GABRA1 ) (Uniprot #: P14867-1) (catalog #: RC205390), β2 subunit ( GABRB2 ) (isoform 2, Uniprot #: P47870-1) (catalog #: RC216424), γ2 subunit ( GABRG2 ) (isoform 2, Uniprot #: P18507-2) (catalog #: RC209260), 5-HT 3 A subunit (catalog #: SC122578, HTR3A , NM_000869), C-terminal FLAG-tagged 5-HT 3 B subunit (catalog #: MR206966, HTR3B , NM_020274), C-terminal FLAG-tagged hERG potassium channel (catalog #: RC215928, KCNH2 , NM_000238, human), and pCMV6 Entry Vector plasmid (pCMV6-EV) (catalog #: PS100001) were obtained from Origene (Rockville, MD, 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 pCMV6 plasmids containing human GABA A receptor α1 subunit ( GABRA1 ) (Uniprot #: P14867-1) (catalog #: RC205390), β2 subunit ( GABRB2 ) (isoform 2, Uniprot #: P47870-1) (catalog #: RC216424), γ2 subunit ( GABRG2 ) (isoform 2, Uniprot #: P18507-2) (catalog #: RC209260), 5-HT 3 A subunit (catalog #: SC122578, HTR3A , NM_000869), C-terminal FLAG-tagged 5-HT 3 B subunit (catalog #: MR206966, HTR3B , NM_020274), C-terminal FLAG-tagged hERG potassium channel (catalog #: RC215928, KCNH2 , NM_000238, human), and pCMV6 Entry Vector plasmid (pCMV6-EV) (catalog #: PS100001) were obtained from Origene (Rockville, MD, 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 pCMV6 plasmids containing human GABA A receptor α1 subunit ( GABRA1 ) (Uniprot #: P14867-1) (catalog #: RC205390), β2 subunit ( GABRB2 ) (isoform 2, Uniprot #: P47870-1) (catalog #: RC216424), γ2 subunit ( GABRG2 ) (isoform 2, Uniprot #: P18507-2) (catalog #: RC209260), 5-HT 3 A subunit (catalog #: SC122578, HTR3A , NM_000869), C-terminal FLAG-tagged 5-HT 3 B subunit (catalog #: MR206966, HTR3B , NM_020274), C-terminal FLAG-tagged hERG potassium channel (catalog #: RC215928, KCNH2 , NM_000238, human), and pCMV6 Entry Vector plasmid (pCMV6-EV) (catalog #: PS100001) were obtained from Origene (Rockville, MD, 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 pCMV6 plasmids containing human GABA A receptor α1 subunit ( GABRA1 ) (Uniprot #: P14867-1) (catalog #: RC205390), β2 subunit ( GABRB2 ) (isoform 2, Uniprot #: P47870-1) (catalog #: RC216424), γ2 subunit ( GABRG2 ) (isoform 2, Uniprot #: P18507-2) (catalog #: RC209260), 5-HT 3 A subunit (catalog #: SC122578, HTR3A , NM_000869), C-terminal FLAG-tagged 5-HT 3 B subunit (catalog #: MR206966, HTR3B , NM_020274), C-terminal FLAG-tagged hERG potassium channel (catalog #: RC215928, KCNH2 , NM_000238, human), and pCMV6 Entry Vector plasmid (pCMV6-EV) (catalog #: PS100001) were obtained from Origene (Rockville, MD, 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 pCMV6 plasmids containing human GABA A receptor α1 subunit ( GABRA1 ) (Uniprot #: P14867-1) (catalog #: RC205390), β2 subunit ( GABRB2 ) (isoform 2, Uniprot #: P47870-1) (catalog #: RC216424), γ2 subunit ( GABRG2 ) (isoform 2, Uniprot #: P18507-2) (catalog #: RC209260), 5-HT 3 A subunit (catalog #: SC122578, HTR3A , NM_000869), C-terminal FLAG-tagged 5-HT 3 B subunit (catalog #: MR206966, HTR3B , NM_020274), C-terminal FLAG-tagged hERG potassium channel (catalog #: RC215928, KCNH2 , NM_000238, human), and pCMV6 Entry Vector plasmid (pCMV6-EV) (catalog #: PS100001) were obtained from Origene (Rockville, MD, USA).

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: The pCMV6 plasmids containing human GABA A receptor α1 subunit ( GABRA1 ) (Uniprot #: P14867-1) (catalog #: RC205390), β2 subunit ( GABRB2 ) (isoform 2, Uniprot #: P47870-1) (catalog #: RC216424), γ2 subunit ( GABRG2 ) (isoform 2, Uniprot #: P18507-2) (catalog #: RC209260), 5-HT 3 A subunit (catalog #: SC122578, HTR3A , NM_000869), C-terminal FLAG-tagged 5-HT 3 B subunit (catalog #: MR206966, HTR3B , NM_020274), C-terminal FLAG-tagged hERG potassium channel (catalog #: RC215928, KCNH2 , NM_000238, human), and pCMV6 Entry Vector plasmid (pCMV6-EV) (catalog #: PS100001) were obtained from Origene (Rockville, MD, USA).

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: The pCMV6 plasmids containing human GABA A receptor α1 subunit ( GABRA1 ) (Uniprot #: P14867-1) (catalog #: RC205390), β2 subunit ( GABRB2 ) (isoform 2, Uniprot #: P47870-1) (catalog #: RC216424), γ2 subunit ( GABRG2 ) (isoform 2, Uniprot #: P18507-2) (catalog #: RC209260), 5-HT 3 A subunit (catalog #: SC122578, HTR3A , NM_000869), C-terminal FLAG-tagged 5-HT 3 B subunit (catalog #: MR206966, HTR3B , NM_020274), C-terminal FLAG-tagged hERG potassium channel (catalog #: RC215928, KCNH2 , NM_000238, human), and pCMV6 Entry Vector plasmid (pCMV6-EV) (catalog #: PS100001) were obtained from Origene (Rockville, MD, USA).

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: The pCMV6 plasmids containing human GABA A receptor α1 subunit ( GABRA1 ) (Uniprot #: P14867-1) (catalog #: RC205390), β2 subunit ( GABRB2 ) (isoform 2, Uniprot #: P47870-1) (catalog #: RC216424), γ2 subunit ( GABRG2 ) (isoform 2, Uniprot #: P18507-2) (catalog #: RC209260), 5-HT 3 A subunit (catalog #: SC122578, HTR3A , NM_000869), C-terminal FLAG-tagged 5-HT 3 B subunit (catalog #: MR206966, HTR3B , NM_020274), C-terminal FLAG-tagged hERG potassium channel (catalog #: RC215928, KCNH2 , NM_000238, human), and pCMV6 Entry Vector plasmid (pCMV6-EV) (catalog #: PS100001) were obtained from Origene (Rockville, MD, 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 pCMV6 plasmids containing human GABA A receptor α1 subunit ( GABRA1 ) (Uniprot #: P14867-1) (catalog #: RC205390), β2 subunit ( GABRB2 ) (isoform 2, Uniprot #: P47870-1) (catalog #: RC216424), γ2 subunit ( GABRG2 ) (isoform 2, Uniprot #: P18507-2) (catalog #: RC209260), 5-HT 3 A subunit (catalog #: SC122578, HTR3A , NM_000869), C-terminal FLAG-tagged 5-HT 3 B subunit (catalog #: MR206966, HTR3B , NM_020274), C-terminal FLAG-tagged hERG potassium channel (catalog #: RC215928, KCNH2 , NM_000238, human), and pCMV6 Entry Vector plasmid (pCMV6-EV) (catalog #: PS100001) were obtained from Origene (Rockville, MD, USA).

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: The pCMV6 plasmids containing human GABA A receptor α1 subunit ( GABRA1 ) (Uniprot #: P14867-1) (catalog #: RC205390), β2 subunit ( GABRB2 ) (isoform 2, Uniprot #: P47870-1) (catalog #: RC216424), γ2 subunit ( GABRG2 ) (isoform 2, Uniprot #: P18507-2) (catalog #: RC209260), 5-HT 3 A subunit (catalog #: SC122578, HTR3A , NM_000869), C-terminal FLAG-tagged 5-HT 3 B subunit (catalog #: MR206966, HTR3B , NM_020274), C-terminal FLAG-tagged hERG potassium channel (catalog #: RC215928, KCNH2 , NM_000238, human), and pCMV6 Entry Vector plasmid (pCMV6-EV) (catalog #: PS100001) were obtained from Origene (Rockville, MD, USA).

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: The pCMV6 plasmids containing human GABA A receptor α1 subunit ( GABRA1 ) (Uniprot #: P14867-1) (catalog #: RC205390), β2 subunit ( GABRB2 ) (isoform 2, Uniprot #: P47870-1) (catalog #: RC216424), γ2 subunit ( GABRG2 ) (isoform 2, Uniprot #: P18507-2) (catalog #: RC209260), 5-HT 3 A subunit (catalog #: SC122578, HTR3A , NM_000869), C-terminal FLAG-tagged 5-HT 3 B subunit (catalog #: MR206966, HTR3B , NM_020274), C-terminal FLAG-tagged hERG potassium channel (catalog #: RC215928, KCNH2 , NM_000238, human), and pCMV6 Entry Vector plasmid (pCMV6-EV) (catalog #: PS100001) were obtained from Origene (Rockville, MD, 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 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: The pCMV6 plasmids containing human GABA A receptor α1 subunit ( GABRA1 ) (Uniprot #: P14867-1) (catalog #: RC205390), β2 subunit ( GABRB2 ) (isoform 2, Uniprot #: P47870-1) (catalog #: RC216424), γ2 subunit ( GABRG2 ) (isoform 2, Uniprot #: P18507-2) (catalog #: RC209260), 5-HT 3 A subunit (catalog #: SC122578, HTR3A , NM_000869), C-terminal FLAG-tagged 5-HT 3 B subunit (catalog #: MR206966, HTR3B , NM_020274), C-terminal FLAG-tagged hERG potassium channel (catalog #: RC215928, KCNH2 , NM_000238, human), and pCMV6 Entry Vector plasmid (pCMV6-EV) (catalog #: PS100001) were obtained from Origene (Rockville, MD, USA).

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: ( 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 pCMV6 plasmids containing human GABA A receptor α1 subunit ( GABRA1 ) (Uniprot #: P14867-1) (catalog #: RC205390), β2 subunit ( GABRB2 ) (isoform 2, Uniprot #: P47870-1) (catalog #: RC216424), γ2 subunit ( GABRG2 ) (isoform 2, Uniprot #: P18507-2) (catalog #: RC209260), 5-HT 3 A subunit (catalog #: SC122578, HTR3A , NM_000869), C-terminal FLAG-tagged 5-HT 3 B subunit (catalog #: MR206966, HTR3B , NM_020274), C-terminal FLAG-tagged hERG potassium channel (catalog #: RC215928, KCNH2 , NM_000238, human), and pCMV6 Entry Vector plasmid (pCMV6-EV) (catalog #: PS100001) were obtained from Origene (Rockville, MD, 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 pCMV6 plasmids containing human GABA A receptor α1 subunit ( GABRA1 ) (Uniprot #: P14867-1) (catalog #: RC205390), β2 subunit ( GABRB2 ) (isoform 2, Uniprot #: P47870-1) (catalog #: RC216424), γ2 subunit ( GABRG2 ) (isoform 2, Uniprot #: P18507-2) (catalog #: RC209260), 5-HT 3 A subunit (catalog #: SC122578, HTR3A , NM_000869), C-terminal FLAG-tagged 5-HT 3 B subunit (catalog #: MR206966, HTR3B , NM_020274), C-terminal FLAG-tagged hERG potassium channel (catalog #: RC215928, KCNH2 , NM_000238, human), and pCMV6 Entry Vector plasmid (pCMV6-EV) (catalog #: PS100001) were obtained from Origene (Rockville, MD, 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: ( A ) HEK293T cells were transfected with NMDA receptor subunits (GluN1 and GluN2A (WT or M705V)) 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. ( B ) HEK293T cells stably expressing FLAG-tagged hERG potassium channels (WT, N470D, or T65P) were transiently transfected with non-targeting (NT) control siRNA or siRNA against SERPINH1 . Forty-eight hours post transfection, cells were lysed, and the total proteins were subjected to SDS-PAGE and immunoblotting with indicated antibodies. The 155 kDa band represents the mature form, and the 135 kDa band represents the immature ER form. Three biological replicates were performed. ( C ) Gaucher patient-derived fibroblasts carrying L444P β-glucocerebrosidase variant were transiently transfected with non-targeting (NT) control siRNA or siRNA against SERPINH1 . Forty-eight hours post transfection, cells were lysed, and the total proteins were subjected to SDS-PAGE and immunoblotting with indicated antibodies. Three biological replicates were performed. Each data point is reported as mean ± SD. Statistical significance was calculated using an unpaired two-tailed Student’s t-Test. * p<0.05, ** p<0.01, *** p<0.001. Figure 7—figure supplement 2—source data 1. Original files for the western blot analysis in . Figure 7—figure supplement 2—source data 2. PDF containing the original blots in with the relevant bands clearly labeled. Figure 7—figure supplement 2—source data 3. Data used for graphs presented in .

Article Snippet: The pCMV6 plasmids containing human GABA A receptor α1 subunit ( GABRA1 ) (Uniprot #: P14867-1) (catalog #: RC205390), β2 subunit ( GABRB2 ) (isoform 2, Uniprot #: P47870-1) (catalog #: RC216424), γ2 subunit ( GABRG2 ) (isoform 2, Uniprot #: P18507-2) (catalog #: RC209260), 5-HT 3 A subunit (catalog #: SC122578, HTR3A , NM_000869), C-terminal FLAG-tagged 5-HT 3 B subunit (catalog #: MR206966, HTR3B , NM_020274), C-terminal FLAG-tagged hERG potassium channel (catalog #: RC215928, KCNH2 , NM_000238, human), and pCMV6 Entry Vector plasmid (pCMV6-EV) (catalog #: PS100001) were obtained from Origene (Rockville, MD, USA).

Techniques: Transfection, Plasmid Preparation, Control, Cell Surface Biotinylation Assay, Western Blot, Membrane, Stable Transfection, Expressing, SDS Page, Derivative Assay, Variant Assay, 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:

Article Snippet: The pCMV6 plasmids containing human GABA A receptor α1 subunit ( GABRA1 ) (Uniprot #: P14867-1) (catalog #: RC205390), β2 subunit ( GABRB2 ) (isoform 2, Uniprot #: P47870-1) (catalog #: RC216424), γ2 subunit ( GABRG2 ) (isoform 2, Uniprot #: P18507-2) (catalog #: RC209260), 5-HT 3 A subunit (catalog #: SC122578, HTR3A , NM_000869), C-terminal FLAG-tagged 5-HT 3 B subunit (catalog #: MR206966, HTR3B , NM_020274), C-terminal FLAG-tagged hERG potassium channel (catalog #: RC215928, KCNH2 , NM_000238, human), and pCMV6 Entry Vector plasmid (pCMV6-EV) (catalog #: PS100001) were obtained from Origene (Rockville, MD, USA).

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

Journal: Annals of Clinical and Translational Neurology

Article Title: A novel de novo GABRA2 gene missense variant causing developmental epileptic encephalopathy in a Chinese patient

doi: 10.1002/acn3.52262

Figure Lengend Snippet: Analysis of GABA A R pentameric structure. (A) A pentameric model featuring 2 α2 subunits (GABRA2), 2 β3 subunits (GABRB3), and 1 γ2 subunit (GABRG2). (B) The structure is rotated 90 degrees for a top‐down view. In the wild‐type GABRA2, Ala308 forms hydrogen bonds with Pro209 of GABRB3 and with another GABRA2 protein at position 238 of GABRG2. (C) In the mutated GABRA2 (p.Ala308Val), the longer valine side chain disrupts these hydrogen bonds, potentially altering structural stability.

Article Snippet: The primary antibodies utilized included: DYKDDDDK Tag (9A3) Mouse mAb (Cat#8146, Cell Signaling Technology, MA, USA, diluted 1:200); GABRB3 Antibody (Cat#PB0627, Boster Biological Technology, Wuhan, China, diluted 1:1000); GABRG2 Antibody (Cat#A02361‐1, Boster Biological Technology, Wuhan, China, diluted 1:1000); and β‐Actin (8H10D10) Mouse mAb (Cat#3700, Cell Signaling Technology, MA, USA, diluted 1:1000).

Techniques:

Journal: Annals of Clinical and Translational Neurology

Article Title: A novel de novo GABRA2 gene missense variant causing developmental epileptic encephalopathy in a Chinese patient

doi: 10.1002/acn3.52262

Figure Lengend Snippet: In vitro functional assay. (A) Western blotting analysis indicated that the GABRA2‐MUT group exhibited protein expression, but it was significantly lower than the GABRA2‐WT group, with a reduction of approximately 70.62% (*** P < 0.001). (B) CO‐IP results showed that wild‐type GABRA2 interacted with GABRB3/GABRG2, while the mutant may disrupt this interaction. The right panel presents normalized CO‐IP analysis, *** P < 0.001.

Article Snippet: The primary antibodies utilized included: DYKDDDDK Tag (9A3) Mouse mAb (Cat#8146, Cell Signaling Technology, MA, USA, diluted 1:200); GABRB3 Antibody (Cat#PB0627, Boster Biological Technology, Wuhan, China, diluted 1:1000); GABRG2 Antibody (Cat#A02361‐1, Boster Biological Technology, Wuhan, China, diluted 1:1000); and β‐Actin (8H10D10) Mouse mAb (Cat#3700, Cell Signaling Technology, MA, USA, diluted 1:1000).

Techniques: In Vitro, Functional Assay, Western Blot, Expressing, Co-Immunoprecipitation Assay, Mutagenesis