Journal: International Journal of Ophthalmology

Article Title: Pirfenidone suppresses the abnormal activation of human Müller cells after platelet-derived growth factor-BB stimulation

doi: 10.18240/ijo.2019.07.04

Figure Lengend Snippet: A: The cells of the normal group and the model group were respectively treated by 0, 0.2 and 0.3 mg/mL pirfenidone. The relative mRNA levels of TGF-β1, TGF-β2 and PEDF were determined by real-time PCR. Results are shown as indicated. The expression levels of TGF-β1, TGF-β2 and PEDF under the indicated treatment of pirfenidone were detected by Western blotting (B) and immunofluorescence (C). Green fluorescence, for the indicated proteins; blue fluorescence for the nucleus. aP<0.05 vs normal+0 mg/mL pirfenidone group, bP<0.01 vs normal+0 mg/mL pirfenidone group, cP<0.0001 vs normal+0 mg/mL pirfenidone group; dP<0.05 vs model+0 mg/mL pirfenidone group, eP<0.01 vs model+0 mg/mL pirfenidone group, fP<0.0001 vs model+0 mg/mL pirfenidone group; gP<0.05 vs model+0.2 mg/mL pirfenidone group, hP<0.01 vs model+0.2 mg/mL pirfenidone group, iP<0.0001 vs model+0.2 mg/mL pirfenidone group.

Article Snippet: The expression of TGF-β1, TGF-β2, PEDF, and β-actin was respectively tested by anti-TGF-β1 antibody (Boster, USA, {"type":"entrez-nucleotide","attrs":{"text":"A04630","term_id":"411029","term_text":"A04630"}} A04630 ), anti-TGF-β2 antibody (Boster, USA, {"type":"entrez-protein","attrs":{"text":"A00892","term_id":"77211","term_text":"pir||A00892"}} A00892 ), anti-PEDF antibody (Abcam, USA, ab233120) and anti-β-actin antibody (Invitrogen, USA, PA1183).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Western Blot, Immunofluorescence, Fluorescence

Journal: Investigative Ophthalmology & Visual Science

Article Title: TGF-β2 Promotes Oxidative Stress in Human Trabecular Meshwork Cells by Selectively Enhancing NADPH Oxidase 4 Expression

doi: 10.1167/iovs.62.4.4

Figure Lengend Snippet: TGF-β2 selectively increases Nox4 mRNA expression in primary human TM cells. Serum-starved confluent cultures of primary TM cells were treated (24 hours) with vehicle (0.1% DMSO) or TGF-β2 (5 ng/mL) and ( A ) relative fold changes in GAPDH-normalized Nox mRNA and p22 phox mRNA isoform expression were quantified by qPCR, as indicated. ( B ) Time-dependent GAPDH-normalized Nox4 mRNA expression in TM cell cultures assayed in the absence (vehicle) or presence of TGF-β2 (5 ng/mL) treated without ( solid circles ) or with actinomycin D (1 µM for 1 hour) prior to ( solid squares ) or at 8 hours post TGF-β2 treatment ( open diamonds ). The continued presence of TGF-β2 ( semi-solid diamonds ) in the presence of actinomycin D did not alter Nox4 mRNA degradation. Data shown in A and B are the means ± SD ( N = 3–5 replicate wells per condition) from separate experiments. *** P < 0.0001; 1-way ANOVA with Tukey's post hoc analysis.

Article Snippet: Serum-starved cultures were treated in the absence (vehicle, 2 µM HCl) or presence of recombinant human TGF-β2 (5 ng/mL; R&D Systems, Inc. [Tocris], Minneapolis, MN, USA, or Cell Signaling Technology, Danvers, MA, USA) for 24 hours unless noted otherwise.

Techniques: Expressing

Journal: Investigative Ophthalmology & Visual Science

Article Title: TGF-β2 Promotes Oxidative Stress in Human Trabecular Meshwork Cells by Selectively Enhancing NADPH Oxidase 4 Expression

doi: 10.1167/iovs.62.4.4

Figure Lengend Snippet: Smad3-knockdown prevents TGF-β2 mediated increases in Nox4 mRNA expression. TM cells were reverse transfected with siRNA (20 nM for 72 hours) against Smad2, Smad3, or scrambled (control), serum starved overnight, and subsequently treated without (vehicle) or with TGF-β2 (5 ng/mL for 24 hours), as indicated. Data shown are the means ± SD ( N = 3 replicate wells per condition) from a single experiment representative of two additional experiments. ** P < 0.001, 1-way ANOVA with Tukey's post hoc analysis. ( A ) GAPDH-normalized Nox4 mRNA expression quantified by qPCR. ( B ) Representative immunoblot of total Smad2 or total Smad3 proteins present in lysates from TM cells reverse transfected with vehicle or siRNA (20 nM) against Smad2 or Smad3 or with control scrambled siRNA (Scr.), as indicated. GAPDH, glyceraldehyde phosphate dehydrogenase protein loading controls.

Article Snippet: Serum-starved cultures were treated in the absence (vehicle, 2 µM HCl) or presence of recombinant human TGF-β2 (5 ng/mL; R&D Systems, Inc. [Tocris], Minneapolis, MN, USA, or Cell Signaling Technology, Danvers, MA, USA) for 24 hours unless noted otherwise.

Techniques: Knockdown, Expressing, Transfection, Control, Western Blot

Journal: Investigative Ophthalmology & Visual Science

Article Title: TGF-β2 Promotes Oxidative Stress in Human Trabecular Meshwork Cells by Selectively Enhancing NADPH Oxidase 4 Expression

doi: 10.1167/iovs.62.4.4

Figure Lengend Snippet: Nox4 protein expression in human TM cells. Serum-starved confluent cultures of primary or transformed TM cells were treated in the absence ( A ) or presence ( B, C ) of vehicle (0.1% DMSO) or TGF-β2 (5 ng/mL for 24 hours), as indicated. A Immunoblots of Nox4 proteins in lysates from quiescent primary or transformed (GTM3) TM cells, as indicated. B and C Nox4 proteins expressed in lysates from primary B or transformed C TM cells treated without (vehicle) or with TGF-β2, as indicated. GAPDH, glyceraldehyde phosphate dehydrogenase protein loading controls. ( Bottom panels ) Densitometric analyses of respective Nox4 protein bands. Data shown are the means ± SD ( N = 3 replicate wells per condition) of GAPDH-normalized Nox4 protein bands from a single experiment representative of two separate experiments. ns, not significant; * P < 0.05; *** P < 0.0001, Student's t-test.

Article Snippet: Serum-starved cultures were treated in the absence (vehicle, 2 µM HCl) or presence of recombinant human TGF-β2 (5 ng/mL; R&D Systems, Inc. [Tocris], Minneapolis, MN, USA, or Cell Signaling Technology, Danvers, MA, USA) for 24 hours unless noted otherwise.

Techniques: Expressing, Transformation Assay, Western Blot

Journal: Investigative Ophthalmology & Visual Science

Article Title: TGF-β2 Promotes Oxidative Stress in Human Trabecular Meshwork Cells by Selectively Enhancing NADPH Oxidase 4 Expression

doi: 10.1167/iovs.62.4.4

Figure Lengend Snippet: A dual inhibitor of Nox1-Nox4 enzyme activity prevents TGF-β2 mediated oxidative stress. Serum-starved confluent cultures of primary TM cells were pre-treated (2 hours) with SB-431542 (10 µM) or GKT137831 (20 µM) and incubated with vehicle (0.1% DMSO) or TGF-β2 (5 ng/mL) for an additional 22 hours, as indicated. ( A ) Representative confocal images of CellROX Green fluorescence in treated cells. Blue , DAPI-stained nuclei. Scale Bar, 100 µm. ( B ) Quantitative comparison of CellROX Green fluorescence. Data shown are the means ± SD ( N = 4 separate wells per condition, 2 fields per well, 15 cells per field). ( C ) Quantitative comparison of DCFDA fluorescence. Data shown are the means ± SD ( N = 8 replicate wells per condition) from a single experiment representative of two separate experiments. ** P < 0.001; *** P < 0.0001, 1-way ANOVA with Tukey's post hoc analysis.

Article Snippet: Serum-starved cultures were treated in the absence (vehicle, 2 µM HCl) or presence of recombinant human TGF-β2 (5 ng/mL; R&D Systems, Inc. [Tocris], Minneapolis, MN, USA, or Cell Signaling Technology, Danvers, MA, USA) for 24 hours unless noted otherwise.

Techniques: Activity Assay, Incubation, Fluorescence, Staining, Comparison

Journal: Investigative Ophthalmology & Visual Science

Article Title: TGF-β2 Promotes Oxidative Stress in Human Trabecular Meshwork Cells by Selectively Enhancing NADPH Oxidase 4 Expression

doi: 10.1167/iovs.62.4.4

Figure Lengend Snippet: A dual inhibitor of Nox1-Nox4 enzyme activity attenuates Smad3-dependent TGF-β2 canonical signaling. Serum-starved confluent cultures of primary TM cells were pretreated (2 hours) with SB-431542 (10 µM) or GKT137831 (20 µM) and incubated with vehicle (0.1% DMSO) or TGF-β2 (5 ng/mL) for an additional 22 hours, as indicated. ( A, B ) Immunoblots and respective densitometric analyses of Smad2 or Smad3 phospho- and total-proteins in lysates from primary TM cells treated without (vehicle) or with TGF-β2, as indicated. Densitometric data shown are the means ± SD ( N = 3 separate experiments) of normalized phospho-Smad protein bands. ( C ) Representative confocal immunofluorescent images of total Smad2 or Smad3 proteins expressed in treated cells. Blue , DAPI-stained nuclei. Scale Bar, 100 µm. ( D, E ) Quantitative comparison of D Smad2 or E Smad3 immunofluorescence. Data shown are the means ± SD ( N = 4–6 separate wells per condition, 2 fields per well, 15 cells per field). * P < 0.05; ** P < 0.001; *** P < 0.0001, 1-way ANOVA with Tukey's post hoc analysis.

Article Snippet: Serum-starved cultures were treated in the absence (vehicle, 2 µM HCl) or presence of recombinant human TGF-β2 (5 ng/mL; R&D Systems, Inc. [Tocris], Minneapolis, MN, USA, or Cell Signaling Technology, Danvers, MA, USA) for 24 hours unless noted otherwise.

Techniques: Activity Assay, Incubation, Western Blot, Staining, Comparison, Immunofluorescence

Journal: Investigative Ophthalmology & Visual Science

Article Title: TGF-β2 Promotes Oxidative Stress in Human Trabecular Meshwork Cells by Selectively Enhancing NADPH Oxidase 4 Expression

doi: 10.1167/iovs.62.4.4

Figure Lengend Snippet: A dual inhibitor of Nox1-Nox4 enzyme activity attenuates TGF-β2 mediated collagen mRNA expression. Serum-starved confluent cultures of ( A – C ) primary or ( D – F ) transformed TM cells were pretreated (2 hours) with SB-431542 (10 µM) or GKT137831 (20 µM) and incubated with vehicle (0.1% DMSO) or TGF-β2 (5 ng/mL) for an additional 22 hours, as indicated. A and D GAPDH-normalized COL1α1 mRNA B and E GAPDH-normalized COL4α1 mRNA ( C and F ) GAPDH-normalized CTGF mRNA expression. Data shown are the means ± SD ( N = 3 replicate wells per condition) representative of two separate experiments A to C or from a single experiment ( D to F ). * P < 0.05; ** P < 0.001; *** P < 0.0001, 1-way ANOVA with Tukey's post hoc analysis.

Article Snippet: Serum-starved cultures were treated in the absence (vehicle, 2 µM HCl) or presence of recombinant human TGF-β2 (5 ng/mL; R&D Systems, Inc. [Tocris], Minneapolis, MN, USA, or Cell Signaling Technology, Danvers, MA, USA) for 24 hours unless noted otherwise.

Techniques: Activity Assay, Expressing, Transformation Assay, Incubation

Journal: Investigative Ophthalmology & Visual Science

Article Title: TGF-β2 Promotes Oxidative Stress in Human Trabecular Meshwork Cells by Selectively Enhancing NADPH Oxidase 4 Expression

doi: 10.1167/iovs.62.4.4

Figure Lengend Snippet: A dual inhibitor of Nox1-Nox4 enzyme activity attenuates TGF-β2 mediated collagen protein expression . Serum-starved confluent cultures of primary TM cells were pretreated (2 hours) with SB-431542 (10 µM) or GKT137831 (20 µM) and incubated with vehicle (0.1% DMSO) or TGF-β2 (5 ng/mL) for an additional 22 hours, as indicated. ( A ) Representative confocal immunofluorescent images of collagens I ( green ) or collagens IV ( red ) proteins expressed in treated cells. Blue , DAPI-stained nuclei. Scale Bar, 100 µm. ( B, C ) Quantitative comparison of B collagen I or C collagen IV immunofluorescence. Data shown are the means ± SD ( N = 4–8 separate wells per condition, 2 fields per well, 15 cells per field). ** P < 0.001; *** P < 0.0001, 1-way ANOVA with Tukey's post hoc analysis.

Article Snippet: Serum-starved cultures were treated in the absence (vehicle, 2 µM HCl) or presence of recombinant human TGF-β2 (5 ng/mL; R&D Systems, Inc. [Tocris], Minneapolis, MN, USA, or Cell Signaling Technology, Danvers, MA, USA) for 24 hours unless noted otherwise.

Techniques: Activity Assay, Expressing, Incubation, Staining, Comparison, Immunofluorescence

Journal: Investigative Ophthalmology & Visual Science

Article Title: TGF-β2 Promotes Oxidative Stress in Human Trabecular Meshwork Cells by Selectively Enhancing NADPH Oxidase 4 Expression

doi: 10.1167/iovs.62.4.4

Figure Lengend Snippet: A dual inhibitor of Nox1-Nox4 enzyme activity attenuates TGF-β2 mediated F-actin stress fiber formation and αSMA expression . Serum-starved confluent cultures of primary TM cells were pretreated (2 hours) with SB-431542 (10 µM) or GKT137831 (20 µM) and incubated with vehicle (0.1% DMSO) or TGF-β2 (5 ng/mL) for an additional 22 hours, as indicated. ( A ) Representative confocal fluorescent images of phalloidin stained F-actin stress fibers ( green ) or immunostained αSMA IV ( red ) proteins expressed in treated cells. Blue , DAPI-stained nuclei. Scale Bar, 100 µm. ( B, C ) Quantitative comparison of B phalloidin fluorescence or C αSMA immunofluorescence ( N = 4–8 separate wells per condition, 2 fields per well, 15 cells per field). *** P < 0.0001, 1-way ANOVA with Tukey's post hoc analysis.

Article Snippet: Serum-starved cultures were treated in the absence (vehicle, 2 µM HCl) or presence of recombinant human TGF-β2 (5 ng/mL; R&D Systems, Inc. [Tocris], Minneapolis, MN, USA, or Cell Signaling Technology, Danvers, MA, USA) for 24 hours unless noted otherwise.

Techniques: Activity Assay, Expressing, Incubation, Staining, Comparison, Fluorescence, Immunofluorescence

Journal: iScience

Article Title: Red blood cell signaling is functionally conserved in Plasmodium invasion

doi: 10.1016/j.isci.2024.111052

Figure Lengend Snippet: Schematic model of RBC cAMP-Ca 2+ signaling during merozoite invasion A functionally conserved RBC cAMP-Ca 2+ signaling crosstalk is manipulated by Plasmodium parasites to facilitate successful invasion. (A) Upon the merozoite release, it attaches and reorients its apical end toward the RBC membrane (I). The binding of species-specific parasite ligands including RH5 in P. falciparum and unknown ligands in P. knowlesi and P. yoelii to their respective host receptors (indicated in right panel) triggers an activation cascade of neighboring RBC molecules in a multimeric membrane complex which contains and activates the β-adrenergic receptors (II). Activation of βAR triggers the dissociation of stimulatory G protein alpha (Gαs) from heterotrimeric G proteins (αβγ subunits) that binds to the transmembrane adenylyl cyclase (III). Activation of transmembrane adenylyl cyclase generates cAMP from ATP via 3′-5′-ATP-cyclization reaction (IV). cAMP molecules bind to the PKA (V) and subsequently activates the kinase. PKA may directly or indirectly be involved in the activation of L-type Ca 2+ channels (VI). (B) PKA may phosphorylate the cytoskeleton proteins to promote overall changes of RBC cytoskeleton structure (VII) which is involved in the insertion of the AMA1-RONs complex (indicated in the rectangle) required for junction formation. (C) Perturbation of the RBC cAMP-Ca 2+ signaling cascade by inhibitors blocks junction formation. “?” represents the unknown RBC membrane proteins. “N” represents nuclei in blue.

Article Snippet: β2-Adrenergic Receptor (D6H2) Rabbit mAb , Cell Signaling Technology , 8513s; RRID: AB_2313733.

Techniques: Membrane, Binding Assay, Activation Assay

Journal: iScience

Article Title: Red blood cell signaling is functionally conserved in Plasmodium invasion

doi: 10.1016/j.isci.2024.111052

Figure Lengend Snippet:

Article Snippet: β2-Adrenergic Receptor (D6H2) Rabbit mAb , Cell Signaling Technology , 8513s; RRID: AB_2313733.

Techniques: Recombinant, Giemsa Stain, Saline, Lysis, Membrane, Fluorescence, Blocking Assay, Purification, Protein Purification, cAMP Assay, Competitive ELISA, Plasmid Preparation, Software, Imaging

Journal: Frontiers in Immunology

Article Title: Latent TGF-β Activation Is a Hallmark of the Tenascin Family

doi: 10.3389/fimmu.2021.613438

Figure Lengend Snippet: The FBG-like domain of the Tenascin family members activates the latent TGF-β in epithelial cells. (A) Western-blot analysis showing P-Smad2, total Smad2/3 and GAPDH levels in NMuMG cultured for 3h onto Non-Coated (N-C) dishes, dishes coated with 22,2 pmol/cm 2 full-length Tenascins (TN) or stimulated with 2 ng/mL TGF-β1, in presence of anti-pan-TGF-β antibody or isotype-matched control IgG (10 μg/mL). (B) Western Blot analysis showing P-Smad2, total Smad2/3 and GAPDH levels in NMuMG cells cultured for 3h in presence of 333 pmol/cm 2 of FBG-like domains as described in (A) . (C) SDS-PAGE analysis of purified recombinant FBG-like domains produced in mammalian cells (FBG) and E. coli (FBG*) resolved on 15% acrylamide gels under reducing conditions (2 µg each). MM, molecular mass markers. (D) Western-Blot analysis of the levels of mature TGF-β1 and LAP(β1) pro-domain associated with 20 pmol of FBG-like domains produced in mammalian cells (FBG) and E coli (FBG*). (E) Western-blot analysis showing P-Smad2, Smad2/3 and GAPDH levels in NMuMG cells stimulated for 1h with active TGF-β1 (2 ng/mL), soluble purified FBG-like domains produced in E Coli (720 nM) or vehicle (PBS).

Article Snippet: For neutralization experiments, mouse monoclonal IgG1 anti-TGF-β1/β2/β3 (Clone 1D11) antibody was purchased from R&D Systems and isotype-matched mouse Gamma Globulin used as negative control from Jackson ImmunoResearch.

Techniques: Western Blot, Cell Culture, Control, SDS Page, Purification, Recombinant, Produced

Journal: Frontiers in Immunology

Article Title: Latent TGF-β Activation Is a Hallmark of the Tenascin Family

doi: 10.3389/fimmu.2021.613438

Figure Lengend Snippet: The FBG-like domains of Tenascins trigger EMT and cytostasis in epithelial cells. (A) F-actin direct fluorescence (red) and E-Cadherin indirect immunofluorescence (green) performed on NMuMG cells cultured for 72h onto N-C dishes, dishes coated with 666 pmol/cm 2 FBG-like domains or stimulated with soluble TGF-β1 (5 ng/mL). Cell nuclei were counterstained with DAPI (blue). Bars, 15 µm. (B) Percentage of cell viability in NMuMG cells cultured for 48h onto N-C dishes, dishes coated with 333, 666 or 999 pmol/cm 2 FBG-like domains, or stimulated with 5 ng/mL TGF-β1. Error bars are means ± SD from 3 independent experiments. * p < 0.05 compared to N-C condition. **** p < 0.0001 compared to N-C condition. ns, not significant. (C) Percentage of cell viability in NMuMG cells cultured for 48h onto N-C dishes, dishes coated with 666 pmol/cm 2 FBG-like domains (FBG-X and FBG-C) or CUB1CUB2 protein fragment (C1C2), or stimulated with 5 ng/mL TGF-β1, in the presence of anti-pan-TGF-β antibody or isotype-matched control IgG (5 μg/mL). Error bars are means ± SD from 3 independent experiments. **, *** and **** respectively correspond to p < 0.01, p < 0.001 and p < 0.0001 compared to N-C condition. # and ### respectively correspond to p < 0.05 and p < 0.001 versus their control IgG-treated counterpart. ns, not significant.

Article Snippet: For neutralization experiments, mouse monoclonal IgG1 anti-TGF-β1/β2/β3 (Clone 1D11) antibody was purchased from R&D Systems and isotype-matched mouse Gamma Globulin used as negative control from Jackson ImmunoResearch.

Techniques: Fluorescence, Immunofluorescence, Cell Culture, 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: bioRxiv

Article Title: Hot-wiring clathrin-mediated endocytosis in human cells

doi: 10.1101/061986

Figure Lengend Snippet: (A) Illustration of chemically-inducible endocytosis. Cells co-express a plasma membrane anchor (CD8-mCherry-FRB) and a clathrin hook (FKBP-Β2-GFP). Rapamycin (200 nM) is added which causes heterodimerization of FKBP and FRB domains. The clathrin hook is rerouted to the plasma membrane. Clathrin recognizes the clathrin hook and the plasma membrane anchor is internalized. (B) Chemically-inducible endocytosis in live cells. Cells expressing CD8-mCherry-FRB with either FKBP-β2-GFP or GFP-FKBP. Stills from live cell imaging experiments are shown, upper image shows the frame before rerouting occurs and lower image is 133 frames (665 s) later. See Supplementary Movies S1 and S2. (C) Example plots to show chemically-inducible endocytosis. Colored traces indicate the total area of bright GFP puncta that form in cells after rerouting. Gray traces show the rerouting of the clathrin hook from the cytoplasm to the plasma membrane (see Methods). (D) Bright green spots formed by chemically-induced endocytosis colocalize with clathrin and contain anti-CD8-Alexa647. Cells co-expressing mCherry-LCa, CD8-dCherry-FRB and either FKBP-β2-GFP or GFP-FKBP, were incubated with anti-CD8-Alexa647 to label the membrane anchor extracellularly, and rapamycin (200 nM) was applied. In B and D, insets show a 2X zoom of the boxed region; closed orange bar indicates rapamycin application; scale bars, 10 μm.

Article Snippet: PDZb-β2-GFP and PDZb1-β2-GFP were made by PCR of PDZb (Addgene #34980) or PDZb1 (Addgene #34981), inserted into FKBP-β2-GFP at XhoI and BamHI, replacing FKBP.

Techniques: Expressing, Live Cell Imaging, Incubation

Journal: bioRxiv

Article Title: Hot-wiring clathrin-mediated endocytosis in human cells

doi: 10.1101/061986

Figure Lengend Snippet: Example confocal live cell imaging of rapamycin (200 nM) application to HeLa cells expressing CD8-mCherry-FRB and FKBP -β 2-GFP. Only the green channel is shown, in grayscale. See . Imaging rate, 0.2 fps. Video speed, 10 fps.

Article Snippet: PDZb-β2-GFP and PDZb1-β2-GFP were made by PCR of PDZb (Addgene #34980) or PDZb1 (Addgene #34981), inserted into FKBP-β2-GFP at XhoI and BamHI, replacing FKBP.

Techniques: Live Cell Imaging, Expressing, Imaging

Journal: bioRxiv

Article Title: Hot-wiring clathrin-mediated endocytosis in human cells

doi: 10.1101/061986

Figure Lengend Snippet: Example confocal live cell imaging of rapamycin (200 nM) application to HeLa cells expressing CD8-mCherry-FRB and GFP-FKBP. Only the green channel is shown, in grayscale. See . Imaging rate, 0.2 fps. Video speed, 10 fps.

Article Snippet: PDZb-β2-GFP and PDZb1-β2-GFP were made by PCR of PDZb (Addgene #34980) or PDZb1 (Addgene #34981), inserted into FKBP-β2-GFP at XhoI and BamHI, replacing FKBP.

Techniques: Live Cell Imaging, Expressing, Imaging

Journal: bioRxiv

Article Title: Hot-wiring clathrin-mediated endocytosis in human cells

doi: 10.1101/061986

Figure Lengend Snippet: (A) Schematic layout of CD8, CD4 and GAP43, used for engineering plasma membrane anchors. (B) Comparison of two other plasma membrane anchors used for chemically-inducible endocytosis. Cells expressed CD4-mCherry-FRB or GAP43-FRB-mRFP with either FKBP-β2-GFP or GFP-FKBP. Stills from live cell imaging experiments are shown, left image shows the frame before rerouting occurs and right image is 133 frames (665 s) later. Both anchors behaved similarly to CD8-mCherry-FRB. Inset shows a 2X zoom of the boxed region. Scale bar, 10 μm.

Article Snippet: PDZb-β2-GFP and PDZb1-β2-GFP were made by PCR of PDZb (Addgene #34980) or PDZb1 (Addgene #34981), inserted into FKBP-β2-GFP at XhoI and BamHI, replacing FKBP.

Techniques: Live Cell Imaging

Journal: bioRxiv

Article Title: Hot-wiring clathrin-mediated endocytosis in human cells

doi: 10.1101/061986

Figure Lengend Snippet: Still from a typical live cell confocal imaging experiment. HeLa cells co-expressing CD8-mCherry-FRB and FKBP-β2-GFP were incubated with anti-CD8-Alexa647 to label the membrane anchor extracellularly, and rapamycin (200 nM) was applied. Insets show 2X zoom of the boxed area. Scale bar, 10 μm.

Article Snippet: PDZb-β2-GFP and PDZb1-β2-GFP were made by PCR of PDZb (Addgene #34980) or PDZb1 (Addgene #34981), inserted into FKBP-β2-GFP at XhoI and BamHI, replacing FKBP.

Techniques: Imaging, Expressing, Incubation

Journal: bioRxiv

Article Title: Hot-wiring clathrin-mediated endocytosis in human cells

doi: 10.1101/061986

Figure Lengend Snippet: Example confocal live cell imaging of rapamycin (200 nM) application to a mitotic HeLa cell expressing CD8-dCherry-FRB, FKBP -β 2-mRuby2 (red) and GFP-LCa (green). Anti-CD8/Alexa647 (blue) was added to label the plasma membrane anchor and then rapamycin was applied. See . Imaging rate, 0.2 fps. Video speed, 10 fps.

Article Snippet: PDZb-β2-GFP and PDZb1-β2-GFP were made by PCR of PDZb (Addgene #34980) or PDZb1 (Addgene #34981), inserted into FKBP-β2-GFP at XhoI and BamHI, replacing FKBP.

Techniques: Live Cell Imaging, Expressing, Imaging

Journal: bioRxiv

Article Title: Hot-wiring clathrin-mediated endocytosis in human cells

doi: 10.1101/061986

Figure Lengend Snippet: Live cell immunolabeling experiments of cells expressing CD8-dCherry-FRB (dark mCherry variant) with FKBP-β2-GFP. Cells were also transfected with siRNAs against GL2 (control) or clathrin heavy chain (CHC). Inhibition of uptake of transferrin-Alexa647 (A647-Tf, blue) was used as a functional test of knockdown efficacy. Scale bar, 10 μm.

Article Snippet: PDZb-β2-GFP and PDZb1-β2-GFP were made by PCR of PDZb (Addgene #34980) or PDZb1 (Addgene #34981), inserted into FKBP-β2-GFP at XhoI and BamHI, replacing FKBP.

Techniques: Immunolabeling, Expressing, Variant Assay, Transfection, Inhibition, Functional Assay

Journal: bioRxiv

Article Title: Hot-wiring clathrin-mediated endocytosis in human cells

doi: 10.1101/061986

Figure Lengend Snippet: Total internal fluorescence microscopy (TIRFM) imaging in RPE1 cells expressing CD8-dCherry-FRB, FKBP-β2-mRuby2 and either dynamin2-GFP (A-C) or GFP-LCa (D-F). Chemically induced endocytosis was triggered by rapamycin (200 nM). (A,D) Kymograph to show the fluorescence along a line in the cell footprint over time. Open and filled orange bars indicate pre and post rapamycin application. (B,E) Two example intensity traces of puncta at the plasma membrane imaged by two-color TIRFM and identified by model-based detection and tracking of FKBP-β2-mRuby2. Plots show for each channel: intensity with a 1 SD uncertainty (shaded area) and background intensity with 2 SD significance threshold (light areas indicate where an event was above threshold, detected). Note the time refers to the event and not to rapamycin application. (C,F) Frame-by-frame montage of the spot analyzed in B and E, respectively. Arrowheads indicate the time when the event was above detection.

Article Snippet: PDZb-β2-GFP and PDZb1-β2-GFP were made by PCR of PDZb (Addgene #34980) or PDZb1 (Addgene #34981), inserted into FKBP-β2-GFP at XhoI and BamHI, replacing FKBP.

Techniques: Fluorescence, Microscopy, Imaging, Expressing

Journal: bioRxiv

Article Title: Hot-wiring clathrin-mediated endocytosis in human cells

doi: 10.1101/061986

Figure Lengend Snippet: Correlative light-electron microscopy to study the uptake of immunolabeled CD8-dCherry-FRB in cells co-expressing either FKBP -β 2-GFP or GFP-FKBP. Results from a single experiment are shown, although CLEM experiments were performed three times. (A) Stills from live cell imaging showing cells sequentially labeled with anti-CD8 and Alexa546 FluoroNanoGold conjugated secondary antibody, before and after addition of rapamycin (200 nM). The same cell was processed for EM and imaged. Scale bars, 10 μm. (B) Electron micrographs of the cells shown in A. Clear uptake of NanoGold into CCVs was seen in cells co-expressing FKBP -β 2-GFP but not GFP-FKBP. Scale bars, 100 nm. (C) Scatter plot of CCV diameters in FKBP -β 2-GFP versus GFP-FKBP samples. Note CCVs in FKBP -β 2-GFP contained NanoGold, whereas GFP-FKBP CCVs did not. Student's t-test, p ± 2.1 × 10 −6 . Bars indicate the mean ± 1 s.d.

Article Snippet: PDZb-β2-GFP and PDZb1-β2-GFP were made by PCR of PDZb (Addgene #34980) or PDZb1 (Addgene #34981), inserted into FKBP-β2-GFP at XhoI and BamHI, replacing FKBP.

Techniques: Electron Microscopy, Immunolabeling, Expressing, Live Cell Imaging, Labeling

Journal: bioRxiv

Article Title: Hot-wiring clathrin-mediated endocytosis in human cells

doi: 10.1101/061986

Figure Lengend Snippet: (A) Orthogonality of chemically-inducible endocytosis. HeLa cells expressing FKBP-β2-GFP alone or together with CD8-mCherry-FRB were tested for ability to internalize transferrin-Alexa647 (A647-Tf) in the presence or absence of rapamycin (200 nM, filled orange bar). (B) Quantification of transferrin uptake and number of GFP-positive puncta in cells. Scatter plot shows values for each cell, bars indicate the mean ± 1 s.d. N exp = 2. Tf, ANOVA, p = 0.13; GFP, ANOVA, p = 5.89 × 10 −6 . (C) Chemically-induced endocytosis can operate independently of endogenous AP-2. Live cell immunolabeling experiments of cells expressing CD8-dCherry-FRB (dark mCherry variant) with FKBP-β2-GFP. Cells were also transfected with siRNAs against GL2 (control) or μ2 subunit of AP-2 complex. Inhibition of uptake of transferrin-Alexa647 was used as a functional test of knockdown efficacy. (D) Quantification of chemically-induced endocytosis. Fraction of puncta that were green and red is shown for each cell (spots), bars indicate the mean ± 1 s.d. N exp = 3. ANOVA, p = 1.48 × 10 −8 . (E) Western blot to assess depletion of μ2 by RNAi. Cells were prepared in parallel with the experiment shown in C. Blotting for μ2 or for GAPDH (as a loading control) is shown. Scale bars, 10 μm.

Article Snippet: PDZb-β2-GFP and PDZb1-β2-GFP were made by PCR of PDZb (Addgene #34980) or PDZb1 (Addgene #34981), inserted into FKBP-β2-GFP at XhoI and BamHI, replacing FKBP.

Techniques: Expressing, Immunolabeling, Variant Assay, Transfection, Inhibition, Functional Assay, Western Blot

Journal: bioRxiv

Article Title: Hot-wiring clathrin-mediated endocytosis in human cells

doi: 10.1101/061986

Figure Lengend Snippet: (A) Stills from typical live cell confocal imaging experiments. HeLa cells expressing CD8-dCherry-FRB, FKBP-β2-mRuby2 and the indicated GFP-tagged construct. Endocytosis was triggered by rapamycin addition (200 nM, filled orange bar). For each, upper image shows the frame before rerouting occurs and lower image is 133 frames (665 s) later. Insets show 2X zoom of the boxed area. Scale bar, 10 μm. (B) Gallery of images to show the spatial relationship between chemically-induced endocytic vesicles (red, FKBP-β2-mRuby2) and GFP-EEA1-positive vesicles (green).

Article Snippet: PDZb-β2-GFP and PDZb1-β2-GFP were made by PCR of PDZb (Addgene #34980) or PDZb1 (Addgene #34981), inserted into FKBP-β2-GFP at XhoI and BamHI, replacing FKBP.

Techniques: Imaging, Expressing, Construct

Journal: bioRxiv

Article Title: Hot-wiring clathrin-mediated endocytosis in human cells

doi: 10.1101/061986

Figure Lengend Snippet: Live cell imaging experiment, mitotic cell expressing CD8-mCherry-FRB with FKBP-β2-GFP. Rapamycin (200 nM) was applied during metaphase. Clear rerouting occurs, but no puncta form in the cytoplasm until after cytokinesis. This cell is shown in Supplementary Movie S3. Scale bars, 10 μm.

Article Snippet: PDZb-β2-GFP and PDZb1-β2-GFP were made by PCR of PDZb (Addgene #34980) or PDZb1 (Addgene #34981), inserted into FKBP-β2-GFP at XhoI and BamHI, replacing FKBP.

Techniques: Live Cell Imaging, Expressing

Journal: bioRxiv

Article Title: Hot-wiring clathrin-mediated endocytosis in human cells

doi: 10.1101/061986

Figure Lengend Snippet: Example confocal live cell imaging of rapamycin (200 nM) application to a mitotic HeLa cell expressing CD8-mCherry-FRB and GFP-FKBP. Green channel is above, in grayscale; red channel is shown below. See . Imaging rate, 0.05 fps. Video speed, 10 fps.

Article Snippet: PDZb-β2-GFP and PDZb1-β2-GFP were made by PCR of PDZb (Addgene #34980) or PDZb1 (Addgene #34981), inserted into FKBP-β2-GFP at XhoI and BamHI, replacing FKBP.

Techniques: Live Cell Imaging, Expressing, Imaging

Journal: bioRxiv

Article Title: Hot-wiring clathrin-mediated endocytosis in human cells

doi: 10.1101/061986

Figure Lengend Snippet: (A) Comparison of four potential clathrin hooks for chemically-inducible endocytosis. Cells expressed CD8-mCherry-FRB with either FKBP-β1-GFP, FKBP-α-GFP, FKBP-epsin-GFP or FKBP-β3-GFP. Stills from live cell imaging experiments are shown, left image shows the frame before rerouting occurs and right image is 133 frames (665 s) later. Inset shows a 2X zoom of the boxed region. Scale bar, 10 m^i. (B) Average plots to show chemically-inducible endocytosis. Colored traces indicate the total area of bright GFP puncta that form in cells after rerouting. Traces were time aligned and averaged. (C) Scatter plot to indicate variability in chemically-induced endocytosis. The total normalized area above threshold at 5 min after rerouting for each cell analyzed is shown (spots). ANOVA, p = 1.142 × 10 −5 . Bars indicate the mean ± 1 s.d. N cell =8–15, N exp =3.

Article Snippet: PDZb-β2-GFP and PDZb1-β2-GFP were made by PCR of PDZb (Addgene #34980) or PDZb1 (Addgene #34981), inserted into FKBP-β2-GFP at XhoI and BamHI, replacing FKBP.

Techniques: Live Cell Imaging

Journal: bioRxiv

Article Title: Hot-wiring clathrin-mediated endocytosis in human cells

doi: 10.1101/061986

Figure Lengend Snippet: (A) Schematic illustration of the putative interactions between β2 and clathrin. Inset shows mutations to delete the clathrin box motif (ΔCBM) or mutation of tyrosine residue Y815 in the appendage (Y-A). Note that Y815 in earlier papers refers to Y829 in our longer isoform. (B) Analysis of clathrin-β2 interaction in vitro. Binding experiments using GST-β2(616–951) wild-type or mutants to pull-down MBP-CHC(1–1074) as indicated. Interaction was assessed by western blotting (i) and a coomassie-stained gel was run in parallel to check for equivalent capture on beads. Three independent experiments were performed and analyzed by densitometry (ii). Bar indicates the mean. (C) Quantification of live-cell imaging experiments to show extent of endocytosis triggered by FKBP-β2-GFP or related mutants (see inset). Colored traces indicate the total area of bright GFP puncta that form in cells after rerouting. Traces were time aligned and averaged. Average traces are shown (i) and a scatter plot (ii) of values at 5 min after rerouting for each cell analyzed is shown (spots). Bars indicate the mean ± 1 s.d. N cell =11–15, N exp =3. ANOVA, p = 7.92 × 10 −5 . Note, the values for FKBP-β2-GFP (WT) and GFP-FKBP (GFP) are also shown in . Example live cell movies are shown in Supplementary , and . (D) Representative images (i) of an immunolabelling experiment to assess endocytosis of CD8 by triggered endocytosis. Scatter plot (ii) to indicate variability in chemically-induced endocytosis. The fraction of green puncta that were also red for each cell analyzed is shown (spots). Bars indicate the mean ± 1 s.d. N cell =8–14, N exp =3. ANOVA, p = 1.66 × 10 −6 .

Article Snippet: PDZb-β2-GFP and PDZb1-β2-GFP were made by PCR of PDZb (Addgene #34980) or PDZb1 (Addgene #34981), inserted into FKBP-β2-GFP at XhoI and BamHI, replacing FKBP.

Techniques: Mutagenesis, In Vitro, Binding Assay, Western Blot, Staining, Live Cell Imaging

Journal: bioRxiv

Article Title: Hot-wiring clathrin-mediated endocytosis in human cells

doi: 10.1101/061986

Figure Lengend Snippet: Example confocal live cell imaging of rapamycin (200 nM) application to HeLa cells expressing CD8-mCherry-FRB and FKBP - 2-GFP(Y-A). Only the green channel is shown, in grayscale. Imaging rate, 0.2 fps. See . Video speed, 10 fps.

Article Snippet: PDZb-β2-GFP and PDZb1-β2-GFP were made by PCR of PDZb (Addgene #34980) or PDZb1 (Addgene #34981), inserted into FKBP-β2-GFP at XhoI and BamHI, replacing FKBP.

Techniques: Live Cell Imaging, Expressing, Imaging

Journal: bioRxiv

Article Title: Hot-wiring clathrin-mediated endocytosis in human cells

doi: 10.1101/061986

Figure Lengend Snippet: Example confocal live cell imaging of rapamycin (200 nM) application to HeLa cells expressing CD8-mCherry-FRB and FKBP -β 2-GFP(ΔCBM). Only the green channel is shown, in grayscale. See . Imaging rate, 0.2 fps. Video speed, 10 fps.

Article Snippet: PDZb-β2-GFP and PDZb1-β2-GFP were made by PCR of PDZb (Addgene #34980) or PDZb1 (Addgene #34981), inserted into FKBP-β2-GFP at XhoI and BamHI, replacing FKBP.

Techniques: Live Cell Imaging, Expressing, Imaging

Journal: bioRxiv

Article Title: Hot-wiring clathrin-mediated endocytosis in human cells

doi: 10.1101/061986

Figure Lengend Snippet: Example confocal live cell imaging of rapamycin (200 nM) application to HeLa cells expressing CD8-mCherry-FRB and FKBP -β 2-GFP(double mutant). Only the green channel is shown, in grayscale. See . Imaging rate, 0.2 fps. Video speed, 10 fps.?

Article Snippet: PDZb-β2-GFP and PDZb1-β2-GFP were made by PCR of PDZb (Addgene #34980) or PDZb1 (Addgene #34981), inserted into FKBP-β2-GFP at XhoI and BamHI, replacing FKBP.

Techniques: Live Cell Imaging, Expressing, Mutagenesis, Imaging