Journal: Journal of Virology

Article Title: Glycosylated NS3/NS3A protein of bluetongue virus facilitates efficient viral egress via lipid raft anchoring

doi: 10.1128/jvi.02144-25

Figure Lengend Snippet: N-linked glycosylation drives plasma membrane accumulation of NS3/NS3A. ( A ) The stability of NS3/NS3A WT and NS3/NS3A N150Q proteins was examined in both transfected (top panels) and BTV-20-infected cells (bottom panels; MOI = 10). For transfection assays, HEK-293T cells were transfected with plasmids expressing NS3/NS3A WT or the N150Q mutant and treated with cycloheximide (CHX; 100 μg/mL) at 18 h post-transfection (designated as 0 h post-CHX treatment) to block de novo protein synthesis. For infection assays, MDOK cells were infected with BTV-20 WT or BTV-20 N150Q and treated with CHX (100 μg/mL) at 10 h post-infection (designated as 0 h post-CHX treatment). Cells were harvested at the indicated time points and analyzed by Western blotting. ( B ) Quantification of NS3/NS3A protein levels shown in panel A was performed by ImageJ densitometric analysis. Protein levels at each time point were normalized to the corresponding 0 h post-CHX treatment (18 h post-transfection or 10 h post-infection, respectively). ( C ) Subcellular localization of NS3/NS3A in MDOK cells infected with BTV-20 WT or BTV-20 N150Q (MOI = 5, 12 h.p.i.). NS3/NS3A (red) was co-stained with ER marker anti-calnexin (green) or Golgi marker anti-syntaxin 6 (green). Fluorescence distribution was evaluated using line-scan intensity profiles. Scale bar, 5 µm. ( D ) Subcellular localization of NS3/NS3A in MDOK cells infected with BTV-20 WT or BTV-20 N150Q (MOI = 5, 12 h.p.i.). NS3/NS3A (red) was co-stained with plasma membrane marker WGA-Alexa Fluor 488 (green). Line-scan intensity profiles are shown. Scale bar, 5 µm. ( E ) Plasma membrane isolation of HEK-293T cells transfected with NS3/NS3A WT or N150Q mutant, followed by Western blot analysis. PM (plasma membrane fraction); NPM (non-plasma membrane fraction); Total (plasma membrane fraction + non-plasma membrane fraction). ( F ) Quantification of NS3/NS3A at the plasma membrane fraction was analyzed by Image J from panel E (* P < 0.05, two-tailed unpaired t-test). ( G ) Confocal imaging of HeLa cells co-transfected with NS3/NS3A (WT or N150Q, red) and VP2 (green) or VP5 (green), showing their subcellular colocalization. Colocalization was assessed by line-scan intensity profiles.

Article Snippet: Commercial antibodies used in this study included anti-FLAG (DYKDDDDK) monoclonal antibody (1:1,000 for immunofluorescence assay [IFA], 1:10,000 for western blotting [WB]; 66008-4-Ig, Proteintech), anti-HA polyclonal antibody (1:100 for IFA, 1:1,000 for WB; 51064-2-AP, Proteintech), anti-β-actin monoclonal antibody (1:10,000 for WB; 66009-1-Ig, Proteintech), anti-Calnexin polyclonal antibody (1:200 for IFA; 10427-2-AP, Proteintech), anti-Syntaxin 6 polyclonal antibody (1:200 for IFA; 10841-1-AP, Proteintech), anti-Flotillin 1 monoclonal antibody (1:100 for IFA; 67968-1-Ig, Proteintech), and anti-Filamin A (FLNA) monoclonal antibody (1:1,000 for WB; 67133-1-Ig, Proteintech).

Techniques: Glycoproteomics, Clinical Proteomics, Membrane, Transfection, Infection, Expressing, Mutagenesis, Blocking Assay, Western Blot, Staining, Marker, Fluorescence, Isolation, Two Tailed Test, Imaging

Journal: medRxiv

Article Title: Genome-wide association study identifies risk variants for sporadic Creutzfeldt-Jakob disease in STX6 and GAL3ST1

doi: 10.1101/2020.04.06.20055376

Figure Lengend Snippet: Illustrating three genome-wide significant loci conferring risk of sCJD (in PRNP, STX6 and GAL3ST1 ) and two genes significant only with gene-wide tests ( PDIA4, BMERB1 ).

Article Snippet: 3.5 μg of pRetroSuper plasmid containing shRNA targeting mouse Stx6 (5’-TGGAATGCTGGAGTGGCAGATCGCTATGG; Origene), Prnp (5’-GAGACAATCTAAACATTCT; as described previously ) or a non-effective scrambled 29mer (5’-GCACTACCAGAGCTAACTCAGATAGTACT; Origene).

Techniques: Genome Wide

Journal: medRxiv

Article Title: Genome-wide association study identifies risk variants for sporadic Creutzfeldt-Jakob disease in STX6 and GAL3ST1

doi: 10.1101/2020.04.06.20055376

Figure Lengend Snippet: CAVIAR utilizes summary statistics and LD structure to predict the probability of each variant being causal, producing a ‘causal set’ with 95% probability of containing the causal SNP, whilst allowing for the possibility of multiple causal SNPs at each locus. Each locus was defined as 100 variants upstream and downstream of the top SNP. Plots show causal posterior probability of each variant at ( a ) PRNP , ( b ) STX6 and ( c ) GAL3ST1 coloured by LD (1000G; EUR) with the top SNP. Circles indicate variants within the 95% causal set.

Article Snippet: 3.5 μg of pRetroSuper plasmid containing shRNA targeting mouse Stx6 (5’-TGGAATGCTGGAGTGGCAGATCGCTATGG; Origene), Prnp (5’-GAGACAATCTAAACATTCT; as described previously ) or a non-effective scrambled 29mer (5’-GCACTACCAGAGCTAACTCAGATAGTACT; Origene).

Techniques: Variant Assay

Journal: medRxiv

Article Title: Genome-wide association study identifies risk variants for sporadic Creutzfeldt-Jakob disease in STX6 and GAL3ST1

doi: 10.1101/2020.04.06.20055376

Figure Lengend Snippet: Plot of –log 10 of P-values from the GWAS association analysis at the STX6 locus (black) and the eQTL association analysis from the GTEx dataset (v7) (red) for: ( a ) STX6 expression in the caudate, ( b ) STX6 expression in the putamen, ( c ) STX6 expression in the hypothalamus, ( d ) KIAA1614 expression in the tibial nerve, ( e ) KIAA1614 expression in the tibial artery. Peaks correspond to the CLPP in the eCAVIAR analysis with a higher degree of colocalisation with increasing CLPP (see Supplementary Table 4).

Article Snippet: 3.5 μg of pRetroSuper plasmid containing shRNA targeting mouse Stx6 (5’-TGGAATGCTGGAGTGGCAGATCGCTATGG; Origene), Prnp (5’-GAGACAATCTAAACATTCT; as described previously ) or a non-effective scrambled 29mer (5’-GCACTACCAGAGCTAACTCAGATAGTACT; Origene).

Techniques: Expressing

Journal: medRxiv

Article Title: Genome-wide association study identifies risk variants for sporadic Creutzfeldt-Jakob disease in STX6 and GAL3ST1

doi: 10.1101/2020.04.06.20055376

Figure Lengend Snippet: N2aPK1/2 cells were transfected with pRetroSuper vectors containing Stx6 (‘shSTX6 1’, ‘shSTX6 2’) or Prnp (‘shPRNP’) targeting shRNAs to generate knockdown cell lines or a scrambled non-silencing (‘shScramble 1’, ‘shScramble 2’) shRNA sequence for controls. Samples were taken prior to plating for SCA for immunoblot (n = 3) and expression normalised to untransfected N2aPK1/2 (indicated by dashed line). ( a-b ) Knockdown of syntaxin-6 protein levels determined by ( a ) immunoblot with anti-syntaxin-6 antibody with ( b ) band intensity measured relative to β-actin loading control (Student’s t-test). ( c-d ) Knockdown of PrP C protein determined by ( c ) immunoblot with anti-PrP antibody ICSM18 with (d) band intensity measured relative to β-actin loading control (Student’s t-test). (e) Average spot count of infected cell number post-4 th split in SCA following infection with RML at 3 × 10 −6 dilution (one-way ANOVA with Tukey’s post-hoc test on log-transformed data). Statistical associations of knockdown lines relative to controls indicated; other results omitted for clarity. All error bars show mean ± SEM; *P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: 3.5 μg of pRetroSuper plasmid containing shRNA targeting mouse Stx6 (5’-TGGAATGCTGGAGTGGCAGATCGCTATGG; Origene), Prnp (5’-GAGACAATCTAAACATTCT; as described previously ) or a non-effective scrambled 29mer (5’-GCACTACCAGAGCTAACTCAGATAGTACT; Origene).

Techniques: Transfection, Knockdown, shRNA, Sequencing, Western Blot, Expressing, Control, Infection, Transformation Assay

Journal: Cellular and Molecular Life Sciences

Article Title: The iRhom homology domain is indispensable for ADAM17-mediated TNFα and EGF receptor ligand release

doi: 10.1007/s00018-021-03845-3

Figure Lengend Snippet: iRhom2 interacts with proteins involved in vesicle-mediated intracellular transport. Volcano plots of the quantitative comparison of a wild-type iRhom2 vs vector GFP (control) and c mutant iRhom2 (W538S) vs vector control (GFP) co-immunoprecipitations from HEK293 cells based on label-free quantification. Significant regulated proteins are labelled orange (requirements: p -value < 0.01, difference/ratio: > fourfold). All proteins belonging to the GO Group “endoplasmic reticulum to Golgi vesicle-mediated transport” (GO:0006888) and syntaxin 6 (STX6) and syntaxin 7 (STX) as well as the already known main iRhom2 interactors are labelled. The volcano plots were generated using Instant Clue . b , d String images of all proteins belonging to the GO Group “endoplasmic reticulum to Golgi vesicle-mediated transport” (GO:0006888) as well as syntaxin 6 (STX6) and syntaxin 7 (STX7) are shown. All these proteins were found in the quantitative comparison of b wild type iRhom2 vs vector control (GFP) and d mutant iRhom2 (W538S) vs vector control (GFP) co-immunoprecipitations based on label-free quantification. (Requirements: p -value < 0.01, difference/ratio: > fourfold). Of note, SEC22A was not found in the interactome screen but identified by western blot ( f ). The images were obtained from String v11.0 (string-db.org) . e – g HEK293 cells stably expressing the indicated HA-tagged iRhom constructs were additionally transfected with myc-tagged syntaxin 6 (STX6_myc) ( e ), syntaxin 10 (STX10_myc) ( f ) or SEC22a_myc ( g ). Co-IP experiments with the different iRhom2 constructs as bait were performed and used for western blotting. To probe for the myc-tagged proteins a α-myc antibody was used. Quantitative analysis binding to iRhom2 can be found in figures S10a, b, c. n = 3

Article Snippet: When indicated cells were transfected with either human STX6 (RC202951; OriGene Technologies), human STX10 (RC215143; OriGene Technologies), human CANX (RC200229; OriGene Technologies) and mouse SEC22a (MR217787; OriGene Technologies).

Techniques: Comparison, Plasmid Preparation, Control, Mutagenesis, Quantitative Proteomics, Generated, Western Blot, Stable Transfection, Expressing, Construct, Transfection, Co-Immunoprecipitation Assay, Binding Assay

Journal: Oncogene

Article Title: Syntaxin 6-mediated Golgi translocation plays an important role in nuclear functions of EGFR through microtubule-dependent trafficking

doi: 10.1038/onc.2013.1

Figure Lengend Snippet: EGF induces translocation of EGFR to the Golgi. (a) HeLa cells were transfected with pDsRed-syntaxin 6. Cells were serum starved overnight and then treated with EGF (50 ng/ml) for 20 min. EGFR was labeled with the indicated antibodies. The boxed areas are shown in detail in the insets. Insets 2–1 and 2–2 show representative colocalizations of EGFR and syntaxin 6. Scale bar, 10 μm. (b) Cells were serum starved overnight and then treated without or with EGF (50 ng/ml) for 20 min. Endogenous EGFR and syntaxin 6 were labeled with a primary antibodies and secondary fluorescein isothiocyanate (donor, green) and Texas-Red (acceptor; red) antibody. An Fc image was obtained using the Zeiss ZEN software. Scale bar, 20 μm. Quantitation of the FRET intensity is shown in the right. (c) Cell lysate was loaded onto the 0–30% OptiPrep density gradient medium and subjected to ultracentrifugation, and fractions were separated using the Gradient Station. The early endosome, the Golgi and ER markers were used to analyze fractions. S, short expose; L, long expose. (d) HeLa cells were treated with or without EGF (50 ng/ml) for 20 min after starvation overnight. The EGFR levels in the Golgi-enriched fraction (fraction 9) were analyzed using immunoblotting. (e) Cells were serum starved overnight and then treated with EGF (50 ng/ml) for 20 min. One cell was used for z-stack scanning. Representative images were shown. The boxed areas are shown in detail in the insets. Scale bar, 10 μm. (f) Cells were transfected with GalNac T2 for 48 h or direct staining of endogenous marker, GM130. Cells were maintained in serum-free media overnight and treated without or with EGF (50 ng/ml) for indicated time and analyzed using confocal microscope. Scale bar, 20 μm. Quantitation of colocalization of EGFR and endosomal markers is shown in the bottom. (g) HeLa cells were transfected with EGFP-GalNac T2. Cells were exposed to serum-free media overnight following treatment without or with EGF (50 ng/ml) for indicated time. Scale bar, 20 μm. The boxed areas are shown in the insets. Quantitation of colocalization of phospho-EGFR and total EGFR with the GalNac T2 is shown in the bottom. (h) HeLa cells were serum-starved overnight before EGF stimulation for indicated time. Total lysate and the Golgi-enriched fractions were performed with sodium dodecyl sulfate–polyacrylamide gel electrophoresis and western blot to examine the phospho-1086 of EGFR and total EGFR levels.

Article Snippet: The syntaxin 6 full-length plasmid was purchased from OriGene (Rockville, MD, USA), which was subcloned into pDsRedC1 (Clontech, Mountain View, CA, USA) for fluorescence staining.

Techniques: Translocation Assay, Transfection, Labeling, Software, Quantitation Assay, Western Blot, Staining, Marker, Microscopy, Polyacrylamide Gel Electrophoresis

Journal: Oncogene

Article Title: Syntaxin 6-mediated Golgi translocation plays an important role in nuclear functions of EGFR through microtubule-dependent trafficking

doi: 10.1038/onc.2013.1

Figure Lengend Snippet: Syntaxin 6 is required for the Golgi translocation of EGFR. (a) Cells were first transfected with syntaxin 6 or control (Ctrl) siRNAs for 24 h and then transfected with GalNac T2 for 48 h. Cells were then maintained in serum-free media overnight and treated without or with EGF (50 ng/ml) for 20 min and analyzed by confocal microscopy. Scale bar, 20 μm. The boxed areas are shown in detail in the insets. Results of quantitation of colocalization of EGFR and Golgi marker are shown in the right panel. (b) Cells were transfected with syntaxin 6 or control siRNAs. After 72 h transfection, cells were maintained in serum-free media overnight and treated without or with EGF (50 ng/ml) for 20 min. The EGFR levels in the Golgi-enriched fraction were analyzed using immunoblotting. (c) Cells were transfected with CCD domain of syntaxin 6 or control vector. After 48 h transfection, cells were maintained in serum-free media overnight and treated without or with EGF (50 ng/ml) for 20 min. Cells were analyzed by confocal microscope. Scale bar, 20 μm. The boxed areas are shown in detail in the insets. Results of quantitation of colocalization of EGFR and Golgi marker are shown in the right panel. (d) Cells were transfected with syntaxin 6 shRNA targeting to the 3′-UTR region or control shRNA. Syntaxin 6 and was restored in cells with knockdown of endogenous syntaxin 6. Cells were maintained in serum-free media overnight and then treated without or with EGF (50 ng/ml) for 20 min. Cellular fractions were subjected to immunoblotting with the indicated antibodies. (e) Cells were transfected with syntaxin 6 or control siRNAs. After 24 h transfection, cells were transfected with GalNac T2 for 48 h. Cells were maintained in serum-free media overnight and treated without or with EGF (50 ng/ml) for 20 min and then analyzed by confocal microscopy. Scale bar, 20 μm. The boxed areas are shown in detail in the insets. Quantitation of colocalization of EGFR and endosomal markers is shown in the right. (f) HeLa cells were serum-starved overnight and stimulated without or with EGF (50 ng/ml) for 20 min. Cell lysates were immunoprecipitated with the indicated antibodies and subjected to immunoblot analysis as indicated. (g) In vitro transcribed and translated biotin-labeled syntaxin 6 was incubated with recombinant GST-fused EGFR fragments, pulled down using glutathione-Sepharose beads and visualized with horseradish peroxidase (HRP) conjugated streptavidin. CT, c-terminal domain; IB, immunoblot; KD, kimase domain fragment; TM, transmembrane domain fragment.

Article Snippet: The syntaxin 6 full-length plasmid was purchased from OriGene (Rockville, MD, USA), which was subcloned into pDsRedC1 (Clontech, Mountain View, CA, USA) for fluorescence staining.

Techniques: Translocation Assay, Transfection, Confocal Microscopy, Quantitation Assay, Marker, Western Blot, Plasmid Preparation, Microscopy, shRNA, Immunoprecipitation, In Vitro, Labeling, Incubation, Recombinant

Journal: Oncogene

Article Title: Syntaxin 6-mediated Golgi translocation plays an important role in nuclear functions of EGFR through microtubule-dependent trafficking

doi: 10.1038/onc.2013.1

Figure Lengend Snippet: Microtubules and dynein are required for EGF-induced Golgi transport of EGFR. (a) Serum-starved cells were treated with EGF. Double staining of EGFR and α-tubulin were subjected to confocal microscopy assay. Scale bars, 20 μm. (b) HeLa cells were transfected with GFP-GalNac T2, treated with microtubules or dynein inhibitors and then stimulated with EGF. The Golgi-enriched fractions were purified and subjected to immunoblot analysis with the indicated antibodies. (c) Serum-starved HeLa cells were treated as shown in (b) and then stimulated with EGF and analyzed by a confocal microscope. Scale bars, 20 μm. The boxed areas are shown in detail in the insets. Representative colocalization of EGFR and GalNac T2 is shown in inset 2–1. Quantitation of cells with Golgi-localized EGFR is shown in the lower panel. (d) HeLa cells were transfected with GFP-GalNac T2 expression plasmid and then transfected with control (ctrl) vector or CDK1 and cyclin B plasmids, respectively. Cells were then serum starved overnight, stimulated with EGF and further analyzed under a confocal microscope. Scale bar, 20 μm. Quantitative results are shown in the right. (e) Representative frames of time-lapse confocal microscopic image of cells treated with or without nocodazole. HeLa cells were transfected with EGFP–EGFR (green) and DsRed–syntaxin 6 (red) plasmids. After serum starvation overnight and EGF stimulation, images were collected at 30-s intervals as indicated. Scale bar, 5 μm. (f) Serum-starved HeLa cells were transfected with dynein shRNAs and then stimulated with EGF. Golgi-enriched fractions were purified and subjected to immunoblot analysis with indicated antibodies. DMSO, dimethyl sulfoxide; Noc, nocodazole; PT, paclitaxel; Van, vanadate.

Article Snippet: The syntaxin 6 full-length plasmid was purchased from OriGene (Rockville, MD, USA), which was subcloned into pDsRedC1 (Clontech, Mountain View, CA, USA) for fluorescence staining.

Techniques: Double Staining, Confocal Microscopy, Transfection, Purification, Western Blot, Microscopy, Quantitation Assay, Expressing, Plasmid Preparation

Journal: Oncogene

Article Title: Syntaxin 6-mediated Golgi translocation plays an important role in nuclear functions of EGFR through microtubule-dependent trafficking

doi: 10.1038/onc.2013.1

Figure Lengend Snippet: Syntaxin 6 is required for EGFR nuclear translocation. (a) HeLa cells were transfected with syntaxin 6 or control siRNAs and maintained in a serum-free media overnight and treated with EGF (50 ng/ml) for 30 min. Quantitation of positive cells with nuclear EGFR is shown in the lower panel. Scale bar, 20 μm. (b) Cells were transfected with syntaxin 6 or control siRNA and maintained in serum-free media overnight and then treated with EGF (50 ng/ml) for 30 min. Cellular fractions were subjected to immunoblotting with the indicated antibodies. (c) Cells were transfected with syntaxin 6 shRNA targeting to the 3′-UTR region or control shRNA. Syntaxin 6 and vector control were restored in cells with knockdown of endogenous syntaxin 6. Cells were maintained in serum-free media overnight and then treated with EGF (50 ng/ml) for 30 min. Cellular fractions were subjected to immunoblotting with the indicated antibodies. (d) HeLa cells were transfected with a control vector and syntaxin 6 CCD and maintained in serum-free media overnight, and then stimulated with EGF. Quantitation of positive cells with nuclear EGFR is shown in the lower panel. Scale bar, 20 μm. (e) HeLa cells were transfected with a control vector and syntaxin 6 CCD and maintained in serum-free media overnight, and then stimulated with EGF. Nuclear and non-nuclear fractions were subjected to immunoblot analysis with the indicated antibodies. DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: The syntaxin 6 full-length plasmid was purchased from OriGene (Rockville, MD, USA), which was subcloned into pDsRedC1 (Clontech, Mountain View, CA, USA) for fluorescence staining.

Techniques: Translocation Assay, Transfection, Quantitation Assay, Western Blot, shRNA, Plasmid Preparation

Journal: Oncogene

Article Title: Syntaxin 6-mediated Golgi translocation plays an important role in nuclear functions of EGFR through microtubule-dependent trafficking

doi: 10.1038/onc.2013.1

Figure Lengend Snippet: Nuclear function of EGFR requires syntaxin 6 and microtubules. (a) After overnight serum starvation, cells were pretreated with the indicated inhibitors for 30-min treatment and then stimulated with EGF for 30 min, followed by chromatin-IP assay. For IgG control, lysate of cells without EGF stimulation was used. (b) Cells were transfected with siRNAs of syntaxin 6. After 72 h transfection, cells were serum starved overnight and then stimulated with EGF for 30 min, followed by chromatin-IP assy. For IgG control, lysate of cells without EGF stimulation was used. (c) Cells were transfected with siRNAs of syntaxin 6. After 72 h transfection, cells were serum starved overnight and then stimulated with EGF for indicated time. Quantitative reverse transcription–polymerase chain reaction (RT–PCR) was used to analyze the mRNA level. (d) HeLa cells transfected with control siRNAs and siRNAs for syntaxin 6 were transfected with reporter plasmids containing CCND1 promoter. Then, after 24 h transfection, cells were maintained in serum-free media overnight and treated with EGF for indicated time. Total lysates were used for luciferase assay. Error bars were derived from three independent experiments. (e) HeLa cells were transfected with control siRNAs and siRNAs for syntaxin 6. After transfection, 4 × 105 cells were seeded in a six-well plate, incubated for 72 h and then counted. (f) HeLa cells were transfected with control siRNAs and siRNAs for syntaxin 6. After 48 h transfection, cells were treated with BrdU (100 μm) for 1 h. Cells were assayed for BrdU incorporation by flow cytometry. (g) BT20 cells were transfected with control siRNAs and siRNAs for syntaxin 6. After 24 h transfection, 2 × 105 cells were seeded in a 12-well plate overnight, treated with 0.1, 1 and 10 μm of gefitinib for 72 h and then counted. (h) OVCAR3 cells were transfected with control siRNAs and siRNAs for syntaxin 6. After 24 h transfection, 2 × 105 cells were seeded in a 12-well plate overnight, treated with 0.1, 1 and 10 μm of gefitinib for 72 h and then counted. (i) A schematic model of syntaxin 6- and microtubule-mediated Golgi and nuclear transport of EGFR.

Article Snippet: The syntaxin 6 full-length plasmid was purchased from OriGene (Rockville, MD, USA), which was subcloned into pDsRedC1 (Clontech, Mountain View, CA, USA) for fluorescence staining.

Techniques: Chromatin Immunoprecipitation, Transfection, Reverse Transcription Polymerase Chain Reaction, Luciferase, Derivative Assay, Incubation, BrdU Incorporation Assay, Flow Cytometry

Journal: Human Molecular Genetics

Article Title: Glucocerebrosidase modulates cognitive and motor activities in murine models of Parkinson’s disease

doi: 10.1093/hmg/ddw124

Figure Lengend Snippet: Effects of partial glucocerebrosidase inhibition on gene expression in the striata of WT and PrP-A53T-SNCA mice

Article Snippet: , Syntaxin6 , Stx6 , 1.00 ± 0.14 , 0.97 ± 0.10 , 0.87 ± 0.05 , 1.05 ± 0.13 , 58244 , Mm01295595_m1.

Techniques: Inhibition, Gene Expression, Saline, Control

Journal: The Journal of Cell Biology

Article Title: CHC22 clathrin mediates traffic from early secretory compartments for human GLUT4 pathway biogenesis

doi: 10.1083/jcb.201812135

Figure Lengend Snippet: HeLa-GLUT4 cells have a functional GLUT4 trafficking pathway that requires CHC22. (A) Representative images of GLUT4 (exofacial HA tag, internal GFP tag) in HeLa-GLUT4 cells before (basal) or after insulin treatment. GLUT4 at the plasma membrane was detected by IF after surface labeling with anti-HA monoclonal antibody (red). Total GLUT4 (green) was detected by GFP tag. Arrows show the GSC. Arrowheads point to peripheral GLUT4 vesicles. Scale bars: 7.5 µm. (B) Left: Representative FACS histogram of surface GLUT4 fluorescence intensities (signal from anti-HA labeling) before (basal) and after insulin treatment (Ins). Middle: Quantification of surface-to-total GLUT4 (HA-to-GFP MFI signals). Data expressed as mean ± SEM, n = 3, 10,000 cells acquired per experiment. Two-tailed unpaired Student’s t test with equal variances: **, P < 0.01. Right: Representative immunoblot for phosphorylated AKT (p-AKT), phosphorylated AS160 (p-AS160), total AS160, and β-actin in HeLa-GLUT4 cells before and after insulin treatment. The migration position of molecular weight (MW) markers is indicated at the left in kilodaltons. (C) Representative images of total GLUT4 (GFP tag, green) and MHCI (blue) before (basal) or after insulin treatment in HeLa-GLUT4 cells transfected with nontargeting control siRNA (siControl) or siRNA targeting CHC22 (siCHC22). Scale bars: 8 µm. (D) Representative FACS histograms of surface GLUT4 fluorescence intensity (signal from anti-HA labeling) in HeLa-GLUT4 cells transfected with siControl or siRNA siCHC22 before (red) or after (blue) treatment with insulin. Histograms are extracted from the experiment quantified in . (E) Representative IF staining for internalized surface-labeled GLUT4 (HA tag, blue) and STX-6 (red) for HeLa-GLUT4 cells at 0, 10, or 30 min after insulin treatment. Total GLUT4 is detected by GFP tag (green). Scale bars: 7.5 µm. (F) Pearson’s overlap quantification for labeling of STX-6 and HA tag. Data expressed as mean ± SEM, n = 3, 14–19 cells per experiment. One-way ANOVA followed by Bonferroni’s multiple comparison post hoc test, ****, P < 0.0001. (G) Left: Representative SIM image of a HeLa-GLUT4 cell stained for STX-6 (red). Total GLUT4 (green) was detected by GFP tag. The gray circle delineates the nucleus (N) and the white square delineates the magnified area displayed in the right image. Scale bars: 10 µm; magnified image: 1 µm. Right: The white dashed line in the magnified area spans the segment for which fluorescence intensities for GLUT4 and STX-6 are plotted below, in green and red, respectively. Arrowheads indicate areas of overlap. (H) Representative IF staining for CHC17 (red) and CHC22 (blue) in HeLa-GLUT4 cells transfected with nontargeting siControl or siRNA targeting CHC17 (siCHC17) or siCHC22, with GLUT4 detected by GFP tag (green). Arrows point to a CHC22-depleted cell. Scale bars: 10 µm for siControl and siCHC17 and 7.5 µm for siCHC22. Merged images in A, C, E, G, and H show red/green overlap in yellow, red/blue overlap in magenta, green/blue overlap in cyan, and red/green/blue overlap in white.

Article Snippet: Commercial sources of antibodies were as follows: rabbit polyclonal anti-CHC17 (ab21679, Abcam), rabbit polyclonal anti-CHC22 antibody (22283-1-AP, Proteintech), mouse monoclonal anti–β-COP (clone maD; G6160, Sigma-Aldrich), rabbit polyclonal anti-IRAP (#3808, Cell Signaling Technology), rabbit monoclonal anti-IRAP (clone D7C5; #6918, Cell Signaling Technology), rabbit polyclonal anti-phospho AKT Ser473 (#9271, Cell Signaling Technology), rabbit polyclonal anti-phospho-AS160 Thr642 (#4288, Cell Signaling Technology), rabbit polyclonal anti-AS160 (#2447, Cell Signaling Technology), goat polyclonal anti-GLUT4 (C-20; sc-1608, Santa Cruz Biotechnologies), rabbit anti-GLUT4 (235003, Synaptic Systems), mouse monoclonal anti-calreticulin (clone FMC75; ADI_SPA-601, Stressgen Bioreagents), sheep polyclonal anti-TGN46 (AHP500G, Bio-Rad), goat polyclonal anti-GM130 (P-20; sc-16268, Santa Cruz Biotechnologies), sheep polyclonal anti-Sec22b (AHP500G, Creative Diagnostics), mouse monoclonal anti-ERGIC-53 (clone 2B10; TA502110, OriGene), rabbit polyclonal anti-ERGIC-53 (E1031, Sigma-Aldrich), rabbit monoclonal anti-LMAN1 (clone EPR6979; ab125006, Abcam), rabbit polyclonal anti-sortilin (ab16640, Abcam), rabbit polyclonal anti-sortilin (12369-1-AP, Proteintech), mouse monoclonal anti-sortilin (clone EPR15010; ab188586, Abcam), mouse monoclonal anti-STX-6 (clone 30/STX-6; AF5664, Becton Dickinson), mouse monoclonal anti–β-actin (clone AC-15; A5441, Sigma-Aldrich), mouse monoclonal anti-HA (clone 16B12; Covance), mouse monoclonal anti-MHCI W6/32 (produced from the hybridoma in the Brodsky laboratory, described in ), goat polyclonal anti-Rab1 (orb153345, Biorbyt), and chicken anti-GFP (A10262, Invitrogen).

Techniques: Functional Assay, Labeling, Fluorescence, Two Tailed Test, Western Blot, Migration, Molecular Weight, Transfection, Staining

Journal: The Journal of Cell Biology

Article Title: CHC22 clathrin mediates traffic from early secretory compartments for human GLUT4 pathway biogenesis

doi: 10.1083/jcb.201812135

Figure Lengend Snippet: The CHC22 compartment is localized proximal to the TGN and does not overlap with the cis-Golgi. (A–C) Representative SIM of the perinuclear region of HeLa-GLUT4 cells (HeLa-G4) and hSkMC-AB1190-GLUT4 (hSkMC) stained for CHC22 (red) and GM130 (A), TGN46 (B), and STX-6 (blue; C). The solid gray lines delineate the nuclear border (N). The dashed white lines span the segment over which fluorescence intensities for GLUT4 (green), CHC22 (red), and GM130, TGN46, and STX-6 (blue) were plotted. Scale bars: 1 µm. In A–C, GLUT4 (green) was detected by GFP tag in HeLa-GLUT4 or immunostained using an anti-GFP antibody in hSkMC-AB1190-GLUT4. Merged images show red/green overlap in yellow, red/blue overlap in magenta, green/blue overlap in cyan, and red/green/blue overlap in white.

Article Snippet: Commercial sources of antibodies were as follows: rabbit polyclonal anti-CHC17 (ab21679, Abcam), rabbit polyclonal anti-CHC22 antibody (22283-1-AP, Proteintech), mouse monoclonal anti–β-COP (clone maD; G6160, Sigma-Aldrich), rabbit polyclonal anti-IRAP (#3808, Cell Signaling Technology), rabbit monoclonal anti-IRAP (clone D7C5; #6918, Cell Signaling Technology), rabbit polyclonal anti-phospho AKT Ser473 (#9271, Cell Signaling Technology), rabbit polyclonal anti-phospho-AS160 Thr642 (#4288, Cell Signaling Technology), rabbit polyclonal anti-AS160 (#2447, Cell Signaling Technology), goat polyclonal anti-GLUT4 (C-20; sc-1608, Santa Cruz Biotechnologies), rabbit anti-GLUT4 (235003, Synaptic Systems), mouse monoclonal anti-calreticulin (clone FMC75; ADI_SPA-601, Stressgen Bioreagents), sheep polyclonal anti-TGN46 (AHP500G, Bio-Rad), goat polyclonal anti-GM130 (P-20; sc-16268, Santa Cruz Biotechnologies), sheep polyclonal anti-Sec22b (AHP500G, Creative Diagnostics), mouse monoclonal anti-ERGIC-53 (clone 2B10; TA502110, OriGene), rabbit polyclonal anti-ERGIC-53 (E1031, Sigma-Aldrich), rabbit monoclonal anti-LMAN1 (clone EPR6979; ab125006, Abcam), rabbit polyclonal anti-sortilin (ab16640, Abcam), rabbit polyclonal anti-sortilin (12369-1-AP, Proteintech), mouse monoclonal anti-sortilin (clone EPR15010; ab188586, Abcam), mouse monoclonal anti-STX-6 (clone 30/STX-6; AF5664, Becton Dickinson), mouse monoclonal anti–β-actin (clone AC-15; A5441, Sigma-Aldrich), mouse monoclonal anti-HA (clone 16B12; Covance), mouse monoclonal anti-MHCI W6/32 (produced from the hybridoma in the Brodsky laboratory, described in ), goat polyclonal anti-Rab1 (orb153345, Biorbyt), and chicken anti-GFP (A10262, Invitrogen).

Techniques: Staining, Fluorescence