Journal: Cell Death & Disease

Article Title: HP1a-mediated heterochromatin formation inhibits high dietary sugar-induced tumor progression

doi: 10.1038/s41419-021-04414-z

Figure Lengend Snippet: A Western blots of protein from the heads of wild type adult flies fed NDS or HDS, using antibodies against H3K9me2, H3, HP1a, or Syntaxin (loading control). B , C Quantification of B H3K9me2 and C HP1a levels from western blots in A , normalized to H3 and Syntaxin, respectively. All results analyzed and presented reflect data from three independent experiments ( n = 90). D Eyes from adult w m4 outcrossed wild-type w 1118 flies fed NDS or HDS for assessment via the PEV assay. E Pigmentation data from the eyes pictured in D , after pigment was extracted and measured at 480 nm. All results analyzed and presented reflect data from three independent experiments ( n = 30). F Third instar eye discs of ras G12V ; csk −/− tumor-bearing flies fed NDS or HDS, with GFP-labeled tumor cells (green) and HP1a immunostaining (red). Scale bar: 100 μm. G Immunofluorescence intensity of HP1a in tumor cells from flies fed HDS compared to those of tumor cells from flies fed NDS. Staining intensity was normalized to that of non-tumor cells in each experimental condition. All results analyzed and presented reflect data from three independent experiments ( n = 15). Results are shown as mean ± SD. Asterisk indicates statistically significant difference (* p < 0.05). GFP green fluorescent protein, HDS high dietary sugar, NDS normal dietary sugar, NS not significant.

Article Snippet: The blotting membrane was probed with primary antibodies: HP1a (C1A9; Developmental Studies Hybridoma Bank), H3K9me2 (07-212; Upstate Biotechnology), H3 (05-928; Millipore), or Syntaxin (8C3; Developmental Studies Hybridoma Bank).

Techniques: Western Blot, Control, Labeling, Immunostaining, Immunofluorescence, Staining

Journal: Journal of cell science

Article Title: Syntaxin 7 contributes to breast cancer cell invasion by promoting invadopodia formation.

doi: 10.1242/jcs.259576

Figure Lengend Snippet: Fig. 5. STX7 interacts with multiple SNARE partners. (A) GBP pulldown was performed with lysates of HeLa cells expressing GFP-vector, and GFP-tagged SNAP23, VAMP2 and VAMP3, pre-treated with 1 mM NEM. Purified GST–GBP (25 µg) was incubated with glutathione–Sepharose beads and allowed to bind with the respective lysates (300 µg) for 5 h at 4°C. Beads were washed, boiled and advanced to immunoblotting using anti-STX7 and anti-GFP antibodies. The number represents the normalized value of prey protein (STX7) with respect to the precipitated protein (GFP-tagged protein) for the blot shown. WCL, whole-cell lysates. (B,C) GBP pulldown was performed with lysates from HEK-293 cells expressing GFP-vector, GFP-tagged VAMP2, STX4 and STX7, pre-treated with 1mM NEM. Purified GST–GBP (25 µg) was incubated with glutathione–Sepharose beads and allowed to bind with the respective lysates from cells expressing GFP-vector (300 µg), GFP–VAMP2 (300 µg), GFP–STX4 (500 µg) or GFP–STX7 (300 µg) for 5 h at 4°C. Beads were washed, boiled and advanced to immunoblotting using anti-STX7, anti-STX2 and anti-GFP antibody. The number represents the normalized value of prey protein with respect to the precipitated protein (GFP-tagged protein) for the blot shown. Results in A–C are representative of three experimental repeats. (D) MDA-MB-231 cells with independent transfection of GFP-tagged VAMP2, VAMP3, VAMP7, SNAP23 or STX4 were immunostained for STX7. Arrowhead indicates colocalized puncta (N=3). Scale bars: 10 μm. (D′) Percentage colocalization was quantified and plotted. N=3, n=140 (GFP–VAMP2), n=92 (GFP–VAMP3), n=154 (GFP–VAMP7), n=100 (GFP–STX4), n=115 (GFP–SNAP23). The data are displayed using SuperPlots; each biological replicate is distinctly color-coded and each dot represents identified percentage colocalization in a field of view (frame), as described in the Materials and Methods, with mean±s.d. N, number of experimental repeats; n, number of cells analyzed.

Article Snippet: Jo u rn al o f Ce ll Sc ie n ce (Millipore, MAB3328), 1:1000 [immunoblotting (IB)], 1:500 (IF); mouse anti-MT1-MMP (R& D Systems, MAB9181-SP), 1:200 (IF); mouse antivinculin (Sigma, V9131), 1:1000 (IB); mouse anti-cortactin (Millipore, 05- 180), 1:1000 (IB), 1:300 (IF); mouse anti-transferrin receptor (Invitrogen, 136800), 1:500 (IF); rabbit anti-actin (Sigma, A2066), 1:2000 (IB); mouse anti-GFP (Roche, 11814460001), 1:2000 (IB); mouse anti-γ-tubulin (Sigma, T6557), 1:3000 (IB); mouse anti-CD63 (DSHB, H5C6), 1:500 (IF); rabbit anti- STX2 (Proteintech, 55033-1-AP), 1:1000 (IB), mouse anti- STX7 (Santa Cruz Biotechnology, sc-514017), 1:1000 (IB), 1:300 (IF); mouse anti-β1-integrin (P5D2, DSHB), 1:1000 (IB), mouse antiphospho-tyrosine (Millipore, 05-1050), 1:1000 (IB); and rabbit anti-Tks5 (Santa Cruz Biotechnology, sc-30122), 1:500 (IF).

Techniques: Expressing, Plasmid Preparation, Purification, Incubation, Western Blot, Transfection

Journal: Journal of cell science

Article Title: Syntaxin 7 contributes to breast cancer cell invasion by promoting invadopodia formation.

doi: 10.1242/jcs.259576

Figure Lengend Snippet: Fig. 7. The proposed model showing that STX7 interacts with multiple SNAREs and forms multiple distinct SNARE complexes. These complexes assist in the fusion of vesicles carrying MT1-MMP to invadopodia, thus, facilitating ECM degradation. However, upon depletion of STX7, trafficking of MT1-MMP is diverted towards the PM rather than invadopodia. Also, silencing of STX7 abrogates the formation of invadopodia, possibly due to hampered trafficking of signaling molecules or growth factors or an unknown cargo carried by STX7 to promote invadopodia formation. Alternatively, when STX4, VAMP2, VAMP3 or STX7–STX4 is depleted, there is reduced MT1-MMP trafficking to cell surface as well as reduced invadopodia formation.

Article Snippet: Jo u rn al o f Ce ll Sc ie n ce (Millipore, MAB3328), 1:1000 [immunoblotting (IB)], 1:500 (IF); mouse anti-MT1-MMP (R& D Systems, MAB9181-SP), 1:200 (IF); mouse antivinculin (Sigma, V9131), 1:1000 (IB); mouse anti-cortactin (Millipore, 05- 180), 1:1000 (IB), 1:300 (IF); mouse anti-transferrin receptor (Invitrogen, 136800), 1:500 (IF); rabbit anti-actin (Sigma, A2066), 1:2000 (IB); mouse anti-GFP (Roche, 11814460001), 1:2000 (IB); mouse anti-γ-tubulin (Sigma, T6557), 1:3000 (IB); mouse anti-CD63 (DSHB, H5C6), 1:500 (IF); rabbit anti- STX2 (Proteintech, 55033-1-AP), 1:1000 (IB), mouse anti- STX7 (Santa Cruz Biotechnology, sc-514017), 1:1000 (IB), 1:300 (IF); mouse anti-β1-integrin (P5D2, DSHB), 1:1000 (IB), mouse antiphospho-tyrosine (Millipore, 05-1050), 1:1000 (IB); and rabbit anti-Tks5 (Santa Cruz Biotechnology, sc-30122), 1:500 (IF).

Techniques:

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: Autophagy

Article Title: BECN2 (beclin 2) Negatively Regulates Inflammasome Sensors Through ATG9A-Dependent but ATG16L1- and LC3-Independent Non-Canonical Autophagy

doi: 10.1080/15548627.2021.1934270

Figure Lengend Snippet: Key resources table

Article Snippet: Anti-STX5 antibody , Cell Signaling Technology , 14151S.

Techniques: Magnetic Beads, Recombinant, Enzyme-linked Immunosorbent Assay, Cloning, Reverse Transcription

Journal: Molecular Biology of the Cell

Article Title: Sec24C/D-isoform–specific sorting of the preassembled ER–Golgi Q-SNARE complex

doi: 10.1091/mbc.E16-04-0229

Figure Lengend Snippet: Syntaxin5 preferentially interacts with Sec24C in its open conformation. (A) Domain organization of human ER-to-Golgi SNARE and fusion proteins used in this study. The H abc domain of Syntaxin5 is depicted in purple, the longin domain of Sec22b in orange, the known IxM motif of Syntaxin5 in red, SNARE motifs in blue, and transmembrane domains (TMD) in green. (B–E) Direct interaction of the ER-to-Golgi SNARE fusion proteins was probed by GST pull-down assay. Sec23A/24A or Sec23A/24C bound to the SNARE-GST fusion proteins indicated and inputs of Sec23A/24A or Sec23A/24C (2.5%) were separated by SDS–PAGE. The lower part of the polyacrylamide gel containing SNARE-GST fusion proteins was stained with CBB, and the upper part was analyzed by Western blotting for the presence of Sec24C (B–D) or Sec23 (E). *N-terminally degraded recombinant Sec24C.

Article Snippet: The following antibodies were used: Sec31A (32/Sec31A, 612350) and GM130 (35/GM130, 610822), both from BD Bioscience (San Jose, CA); calnexin (ab75801) and Sec22L (ab116676), both from Abcam (Cambridge, MA); Sec23 (E19, sc-12107), Sec24A (N-14, sc-169279), ERGIC53 (C-6, sc-365158), Syntaxin5 (B-8, sc-365124), GS27 (25, sc-135932), Sec22b (29-F7, sc-101267), and Bet1 (17, sc-136390), all from Santa Cruz Biotechnology (Dallas, TX); and Sec24C (SZ505), a kind gift from Randy Schekman (University of California, Berkeley, CA).

Techniques: Pull Down Assay, SDS Page, Staining, Western Blot, Recombinant

Journal: Molecular Biology of the Cell

Article Title: Sec24C/D-isoform–specific sorting of the preassembled ER–Golgi Q-SNARE complex

doi: 10.1091/mbc.E16-04-0229

Figure Lengend Snippet: Mutation of the IxM-binding site within Sec24C affects sorting of the three ER–Golgi Q-SNAREs Syntaxin5, GS27, and Bet1. (A) Recombinant purified inner coat subcomplex His6-Sec23A/24C wild type or His6-Sec23A/24C LIL895AAA (1 μg) expressed in Sf9 insect cells was separated by SDS–PAGE and stained with CBB. (B) Formation of COPII vesicles was reconstituted in vitro by incubation of semi-intact HeLa cells with Sar1b, Sec23A/24C wild type, or Sec23A/24C LIL895AAA or Sec13/31A in the presence of GTP and an ATP-regenerating system (ATPr) as indicated. Newly formed vesicles were separated from donor membranes by differential centrifugation. The vesicle fractions and 5% of SICs used for reconstitution were analyzed by Western blotting for the presence of the non–vesicle membrane marker calnexin and the ER-to-Golgi Q-SNARE proteins Syntaxin5, GS27, and Bet1. (C) Amount of vesicle membrane marker ERGIC53 and ER-to-Golgi Q-SNARE proteins Syntaxin5, GS27, and Bet1 in vesicle fractions quantified using Li-COR Image Studio software ( n = 3; horizontal lines indicate the means). The lower band of Syntaxin5 (isoform 2, marked by an asterisk) was used for quantification.

Article Snippet: The following antibodies were used: Sec31A (32/Sec31A, 612350) and GM130 (35/GM130, 610822), both from BD Bioscience (San Jose, CA); calnexin (ab75801) and Sec22L (ab116676), both from Abcam (Cambridge, MA); Sec23 (E19, sc-12107), Sec24A (N-14, sc-169279), ERGIC53 (C-6, sc-365158), Syntaxin5 (B-8, sc-365124), GS27 (25, sc-135932), Sec22b (29-F7, sc-101267), and Bet1 (17, sc-136390), all from Santa Cruz Biotechnology (Dallas, TX); and Sec24C (SZ505), a kind gift from Randy Schekman (University of California, Berkeley, CA).

Techniques: Mutagenesis, Binding Assay, Recombinant, Purification, SDS Page, Staining, In Vitro, Incubation, Centrifugation, Western Blot, Membrane, Marker, Software

Journal: Molecular Biology of the Cell

Article Title: Sec24C/D-isoform–specific sorting of the preassembled ER–Golgi Q-SNARE complex

doi: 10.1091/mbc.E16-04-0229

Figure Lengend Snippet: Model for Sec24 isoform–dependent sorting into COPII vesicles of the preassembled ER-to-Golgi Q-SNARE complex Sytaxin5/GS27/Bet1. On membrane recruitment of the inner coat subcomplex Sec23/Sec24 via the small GTP-binding protein Sar1, the Sec24 subunit binds to either the R-SNARE Sec22b (Sec24A/B) via a conformational epitope or the Q-SNARE complex Syntaxin5/GS27/Bet1 (Sec24C/D). The ER-to-Golgi Q-SNARE complex binds to a conserved binding site within Sec24C or Sec24D via the motif IxM present in the flexible linker region between the H abc domain and the SNARE motif of its subunit Syntaxin5. The different Sec24 isoforms are not segregated to distinct ERES, and hence their cargo proteins are present on the same vesicle membranes.

Article Snippet: The following antibodies were used: Sec31A (32/Sec31A, 612350) and GM130 (35/GM130, 610822), both from BD Bioscience (San Jose, CA); calnexin (ab75801) and Sec22L (ab116676), both from Abcam (Cambridge, MA); Sec23 (E19, sc-12107), Sec24A (N-14, sc-169279), ERGIC53 (C-6, sc-365158), Syntaxin5 (B-8, sc-365124), GS27 (25, sc-135932), Sec22b (29-F7, sc-101267), and Bet1 (17, sc-136390), all from Santa Cruz Biotechnology (Dallas, TX); and Sec24C (SZ505), a kind gift from Randy Schekman (University of California, Berkeley, CA).

Techniques: Membrane, Binding Assay

Journal: ACS chemical neuroscience

Article Title: Chronic Social Isolation Stress during Peri-Adolescence Alters Presynaptic Dopamine Terminal Dynamics via Augmentation in Accumbal Dopamine Availability

doi: 10.1021/acschemneuro.8b00360

Figure Lengend Snippet: Assessment of several terminal protein expression levels. Relative protein expression levels of (A) VMAT2 (n = 7 in both groups), (B) Synaptigyrin-3 (aGH: n = 6; aSI: n = 5), (C) Syntaxin-1 (aGH: n = 7; aSI: n = 6), and (D) Munc13-3 (aGH: n = 8; aSI: n = 8) were measured. None of these proteins had different expression levels in aGH and aSI rats. (insets) The representative Western blot images, with the respective protein and actin for comparison. Group housed, aGH, blue; Socially isolated, aSI, red.

Article Snippet: Subsequently, blots were incubated with agitation for 2 h at room temperature in TBS-T/5% bovine serum albumin (05470; Sigma-Aldrich) solution containing the following primary antibody concentrations: VAMT2 (1:2000; AB1598P; Millipore Sigma); Synaptogyrin-3 (1:1000; ab106460; abcam); Syntaxin-1 (1:1000; ANR-002; Alomone laboratories).

Techniques: Expressing, Western Blot, Isolation