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: 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: Plasmid Preparation, Mutagenesis, Generated, Western Blot, Stable Transfection, Expressing, Construct, Transfection, Co-Immunoprecipitation Assay, Binding Assay

Journal: Traffic (Copenhagen, Denmark)

Article Title: Copper Directs ATP7B to the Apical Domain of Hepatic Cells via Basolateral Endosomes

doi: 10.1111/tra.12229

Figure Lengend Snippet: A-C) WIF-B cells incubated overnight in 10 μM BCS to stage endogenous ATP7B at the TGN region. D-F) Cells switched to 10 μM CuCl2 for 60 minutes. G-I) Cu-treated cells that were rinsed and re-incubated in 10 μM BCS for 180 minutes. After fixation, cells were triple-stained with antibodies to ATP7B (green), Syntaxin 6, a post-TGN marker (red) and aminopeptidase N, APN, an apical surface marker (blue). Single confocal planes are shown. J) The fraction of total ATP7B fluorescence that localized to the TGN or apical region in each condition was quantified as the extent of overlap with Syntaxin 6 or APN, respectively. Data shown represent the mean +/- SEM from at least 3 confocal stacks (approximately 28 WIF-B cells/stack, obtained from a single experiment).

Article Snippet: Primary antibodies used for indirect immunofluorescence were as follows: for , , S5 , and 6 we used rat anti-ATP7B (S. Lutsenko, Johns Hopkins School of Medicine); For , S3 , S4 , 5 , 7 & 8 we used rabbit anti-ATP7B (#ab124973, Abcam, Cambridge, MA); for and S1 we used rabbit anti-ATP7B (Dr. J. Gitlin, Brown Alpert Medical School, Providence, RI); rabbit anti-aminopeptidase N (APN, #1637, ( 22 ) guinea pig anti-dipeptidyl-peptidase 4 (DPP4, ( 41 )) goat anti-EEA1 (#sc-6415, Santa Cruz Biotechnology, Santa Cruz, CA); mouse anti-TGN38, and -Syntaxin 6 (BD Biosciences, San Jose, CA); mouse anti-LAMP1 (#H4A3-s, Developmental Studies Hybridoma Bank, Iowa City, IA); rabbit anti-Cathepsin D (Dr A. Hasilik, Marburg, Germany); rabbit anti-LC3B (#2775, Cell Signaling Technology, Danvers, MA).

Techniques: Incubation, Staining, Marker, Fluorescence

Journal: Gene Expression

Article Title: Molecular Cloning of the m- Golsyn Gene and its Expression in the Mouse Brain

doi:

Figure Lengend Snippet: Subcellular distribution of m-Golsyn protein in mouse cerebral cortex. (A) Procedure for subcellular fractionation of mouse cerebral cortex. Homogenates were separated into fractions enriched in nuclei (N), 100,000 × g supernatant (S), 100,000 × g precipitate (P), synaptic plasma membrane (SM), presynaptic cytosol (PC), and synaptic vesicle (SV) by differential centrifugation. (B) Fractions were prepared as shown in (A), and an aliquot (20 μg protein) of each of these fractions was resolved by 7.5% SDS-PAGE and subjected to immunoblot analysis with antibodies against GOLSYN, synaptophysin, syntaxin 6, or PDI. Synaptophysin, syntaxin 6, and PDI were used as markers for synaptic vesicle, Golgi apparatus, and endoplasmic reticulum, respectively. Solid and gray arrowheads indicate the position of m-Golsyn and PDI, respectively. (C) P and SV fractions, prepared as shown in (A), were fractionated by ultracentrifugation of a sucrose gradient and subjected to immunoblot analysis.

Article Snippet: The following antibodies were purchased from the sources indicated: mouse anti-protein disulfide isomerase (PDI) antibody, from StressGen Biotechnologies (Canada, BC); mouse anti-syntaxin 6 antibody, from BD Transduction (San Diego, CA); mouse anti-synaptophysin antibody and mouse anti-β-tubulin antibody, from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); mouse anti-neuronal nuclei (NeuN) monoclonal antibody, from Chemicon International, Inc. (Temecula, CA); mouse monoclonal anti-glial fibrillary acidic protein (GFAP) antibody and FITC-conjugated anti-mouse IgG antibody, from Sigma Chemical Co. (St. Louis, MO); horseradish peroxidase (HRP)-conjugated, swine anti-rabbit immunoglobulins, from DakoCytomation Inc. (Car-pinteria, CA); peroxidase-linked anti-mouse Ig, from Amersham Biosciences Corp. (Piscataway, NJ); biotinylated anti-rabbit IgG antibody, from Vector Laboratories Inc. (Burlingame, CA); Texas Red-conjugated goat anti-rabbit IgG, from Molecular Probes Inc. (Eugene, OR).

Techniques: Fractionation, Centrifugation, SDS Page, Western Blot

Journal: Molecular Brain

Article Title: Mice with cleavage-resistant N-cadherin exhibit synapse anomaly in the hippocampus and outperformance in spatial learning tasks

doi: 10.1186/s13041-021-00738-1

Figure Lengend Snippet: Protein profiles of GD mouse brain is normal but for the absence of CTF1. a Representative immunoblot of endogenous proteins related to glutamatergic synapse and/or N-cadherin in the total brain lysate and a synaptosomal fraction from GD and WT mice. No recognizable difference was found in syntaxin 6, PSD95, synaptophysin, full-length N-cadherin, phospho-GluA1, GluA1, GluA2, GLT-1, phospho-AKT, AKT, α-tubulin). b Immunoblot detection of endogenous N-cadherin with an antibody against a C-terminal region in the total lysates of primary cultured cerebrocortical neurons from WT and GD mice. While the full-length form (FL, 130 kDa) was detected both in WT and GD samples, the cytoplasmic fragment CTF1 (45 kDa) was detected only in those from WT, but not in those from GD mice (left). Similar results were obtained from the synaptosomal fraction (right). Non-specific bands appeared around 50 kDa (*) and above

Article Snippet: We used commercial antibodies for the following antigens: N-cadherin (clone 32) and syntaxin-6 (clone 30, BD); β-actin, PSD95, and HA (Sigma); synaptophysin (clone SY38, Abcam); ADAM10 (AB19026), Presenillin1 (MAB5232), GluA1 (MAB2263), and GLT1 (MAB2262, Chemicon/Millipore); GluA2 (#13607), AKT (#9272), phospho-AKT (#9271), and α-Tubulin (#2144, CST); mouse and rabbit IgG, HRP-conjugated (NA931V and NA931934, GE Healthcare).

Techniques: Western Blot, Cell Culture

Journal:

Article Title: Secretory Granule Membrane Protein Recycles Through Multivesicular Bodies

doi: 10.1111/j.1600-0854.2010.01066.x

Figure Lengend Snippet: (A) The PAM-1 epitope recognized by the antibody used for these studies is indicated (Ab). Cleavage to PHMs and PALm (both of which are detected by this antibody) occurs in SGs; PAMs, TMD/CD and a soluble fragment of the cytosolic domain (sf-CD) are shown. (B) The steady state distribution of PAM was compared to that of ACTH, TGN38, syntaxin 6, EEA1 and CI-MPR. Scale bar, 10 μm (C) EM detection of PAM in cryosections using PAM antibody followed by protein A-10 nm gold shows PAM in the TGN, in MVB intralumenal vesicles and in SGs; G = Golgi cisternae; TGN, trans-Golgi network. Scale bar, 200 nm. (D) The paradigm used to evaluate the return of PAM-1 biotinylated on the cell surface to SGs is illustrated; nine wells of PAM-1 AtT-20 cells were exposed to membrane impermeant activated biotin (Methods) for 10 min at 37°C; after the reaction was quenched, cells were harvested immediately (Pulse; n = 3) or chased for 60 min. After 0-60 min, medium was harvested (n = 6), triplicate wells were incubated in Basal or Stim (2 mM BaCl2, 1 μM phorbol myristate acetate; PMA) media for 30 min. Media and Cells were harvested for analysis. Biotinylated PAM proteins recovered from Cells and Media were visualized by Western blot; molecular weight marker mobilities are shown to the right. NB, no biotin control and -, no sample in lane. Boxes show typical regions quantified and asterisk marks nonspecific background. Medium lanes received 16 times more sample than Pulse. Quantification: data for secreted biotinylated PAM proteins are expressed relative to the amount of biotinylated PAM-1 in cell lysates after the Pulse.

Article Snippet: Monoclonal syntaxin 6 antibody was from BD Transduction Laboratories.

Techniques: Incubation, Western Blot, Molecular Weight, Marker

Journal:

Article Title: Secretory Granule Membrane Protein Recycles Through Multivesicular Bodies

doi: 10.1111/j.1600-0854.2010.01066.x

Figure Lengend Snippet: (A) AtT-20 PAM-1 cells were allowed to internalize PAM antibody (red) for 5-60 min. Immunostaining for syntaxin 6 (green) identifies the TGN, immature SGs and endosomes (23-25), compartments occupied by PAM at steady state. Colocalization of internalized antibody with syntaxin 6 was first detectable at 10 min, and increased markedly at 20 and 40 min; scale bar, 10 μm. (B) Focal colocalization of PAM antibody internalized for 40 min (green) and chromogranin A (red) in the TGN. Scale bar, 10 μm. (C) PAM-1 antibody was internalized for 1 h; cells were then fixed and prepared for cryosectioning. PAM-1 antibody was detected with 10 nm protein A-gold (arrowhead). After blocking, the sections were incubated with ACTH antibody that was detected with 15 nm protein A-gold. Scale bar, 200 nm. (D-F) PAM antibody internalized for 60 min at 37°C was visualized by pre-embedding staining. Label was seen in MVBs, in the TGN and in immature SGs (arrows). An MVB (F) is shown after more mild silver intensification of the gold label, allowing better resolution of the ultrastructure and distinction from immature SGs. Scale bars, 200 nm. (G) Cationized ferritin internalized for 1 h is seen as a fine granular label in MVBs and immature SGs. The insert shows cationized ferritin in the TGN and in the membrane enclosing an early condensation of secretory material in the TGN (arrows). Scale bar, 200 nm.

Article Snippet: Monoclonal syntaxin 6 antibody was from BD Transduction Laboratories.

Techniques: Immunostaining, Blocking Assay, Incubation, Staining

Journal:

Article Title: Secretory Granule Membrane Protein Recycles Through Multivesicular Bodies

doi: 10.1111/j.1600-0854.2010.01066.x

Figure Lengend Snippet: (A) After uptake for 1 h at 20°C, PAM-1/Ab complexes (red) accumulated in rounded endosomes of varying size; no label was seen in the TGN (syntaxin 6 staining, green, compare to Fig. 3A). (B,C) Using PAM Ab/protein A-gold complexes under the same conditions, these structures were identified as MVBs; the proportion of gold particles associated with the limiting (Ext) versus internal (Int) vesicle membrane was quantified (± SEM of two separate experiments) (D). Scale bars: A, 5 μm; B,C, 100 nm.

Article Snippet: Monoclonal syntaxin 6 antibody was from BD Transduction Laboratories.

Techniques: Staining