human stx6 rc202951  (OriGene)

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