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

Journal: The Journal of Biological Chemistry

Article Title: Limited proteolysis as a tool to probe the tertiary conformation of dysferlin and structural consequences of patient missense variant L344P

doi: 10.1074/jbc.M117.790014

Figure Lengend Snippet: Sucrose density ultracentrifugation is consistent with C2A as a solitary domain with evidence for multimerization of midregion and mini-dysferlinC72 modules. A, primary human myotubes differentiated for 4 days were harvested via scrape injury, solubilized in lysis buffer, and sedimented on a 5–20% linear sucrose gradient. 13 fractions were removed from top (5%) to bottom (20%) and analyzed via SDS-PAGE. Western blots were probed with dysferlin N- and C-terminal antibodies and syntaxin-4. Mouse brain lysate (strain A/J) and human serum were also run on a separate 5–20% gradient and stained with GelCode or the membrane was probed with syntaxin-1 for known sedimentation standards. The graph shows quantification of sedimentation profiles for dysferlin (blue), mini-dysferlinC72 (mDF) (aqua), and known standards albumin (4S), IgG (7S), and fibrinogen (9S). Full-length (FL) dysferlin is detected in all of the dense fractions of the gradient; in contrast, mini-dysferlinC72 shows a clear sedimentation peak in fraction 4, consistent with formation of a discreet oligomeric species. B, C-terminal regions of dysferlin mediate oligomerization, whereas the C2A domain appears to exist as a monomeric species. HEK293 cells transiently transfected with DYSF40a were harvested via scrape injury 24 h post-transfection and subjected to limited proteolysis with 125 μg/ml trypsin and sucrose gradient ultracentrifugation. A total of 13 fractions were removed from top (5%) to bottom (20%) and analyzed via SDS-PAGE. Romeo displayed peak sedimentation of the excised C2A domain in fractions 2 and 3, similar to sedimentation of syntaxin-4 (34 kDa; see Fig. 4A) and broadly consistent with migration as a monomeric species. Midregion fragments detected by Hamlet-2 and FerA antibodies show clear sedimentation peaks in fractions 5 and 6, similar to IgG (150–180 kDa; 7S). C-terminal tryptic fragments were reproducibly difficult to detect, often with smeared banding, suggesting that once extracted from membrane bilayers into solution they may be susceptible to proteolysis.

Article Snippet: Antibodies used included Romeo (α-rabbit, 1:1000; Abcam {"type":"entrez-nucleotide","attrs":{"text":"Ab124684","term_id":"55166618","term_text":"AB124684"}} Ab124684 ), Hamlet-2 (α-mouse, 1:500; Leica Microsystems HAMLET-2-CE), anti-Fer-A (α-rabbit, 1:1000; Abcam HPA021945), anti-C2DE (α-rabbit, 1:500; Sigma-Aldrich SAB2100636), Hamlet-1 (α-mouse, 1:5000; Leica Microsystems HAMLET-CE), anti-Myc 9E10 (α-mouse, 1:1000; Santa Cruz Biotechnology SC-40), anti-Myc (α-rabbit, 1:5000; Abcam Ab9106), anti-GAPDH (α-mouse, 1:10,000; Merck Millipore MAB374), anti-syntaxin-4 (α-rabbit, 1:250; Synaptic Systems 110042), anti-syntaxin-1 (α-mouse, 1:250; Synaptic Systems 110011), anti-myosin MY32 (1:10,000; Sigma-Aldrich M4276), spectrin (1:100; Leica Biosystems SPEC1-CE), and α-actinin-2 4A3 (1:300,000; a gift from A. Beggs, Children's Hospital Boston, Boston, MA).

Techniques: Lysis, SDS Page, Western Blot, Staining, Sedimentation, Transfection, Migration

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: PLoS Biology

Article Title: Neuron Specific Rab4 Effector GRASP-1 Coordinates Membrane Specialization and Maturation of Recycling Endosomes

doi: 10.1371/journal.pbio.1000283

Figure Lengend Snippet: Immunogold EM of hippocampal neurons labeled with 10 nm protein A gold for Rab4 and with 15 nm protein A gold for GRASP-1 (A), with 10 nm protein A gold for syntaxin 13 and with 15 nm protein A gold for GRASP-1 (B), with 10 nm protein A gold for syntaxin 13 and with 15 nm protein A gold for Rab4 (C), or with 15 nm protein A gold for GRASP-1, with 5 nm protein gold for syntaxin 13, and with 10 nm protein A gold for rab4 (D). Arrow denotes tubular endosomal membrane to which GRASP-1, syntaxin 13, and Rab4 localized. EE indicates early endosomes and scale bar is 100 nm.

Article Snippet: The following antibodies were obtained from commercial sources: rabbit anti-GRASP-1 (AB96361), mouse anti-β-actin, mouse anti-GluR2 (Chemicon), mouse anti-Rab4, mouse anti-EEA1 (BD Biosciences), mouse anti-FLAG, mouse anti-MAP2, mouse anti-αtubulin (Sigma), mouse anti-GFP (Roche), mouse anti-bassoon (Stressgen), rabbit anti-β-galactosidase (MP Biomedicals), mouse anti-β-galactosidase (Promega), rabbit anti-GluR1 (Calbiochem), mouse anti-HA (Roche), rabbit anti-myc (Upstate Biotechnology), mouse LAMP-1 (Stressgen), mouse anti-myc, rabbit anti-Rab5 (Santa Cruz Biotechnology), rabbit anti-syntaxin 13 (Synaptic Systems), human anti-EEA1, and mouse anti-human TfR (ATCC).

Techniques: Labeling

Journal: PLoS Biology

Article Title: Neuron Specific Rab4 Effector GRASP-1 Coordinates Membrane Specialization and Maturation of Recycling Endosomes

doi: 10.1371/journal.pbio.1000283

Figure Lengend Snippet: (A) Lysates of COS-7 cells cotransfected with GFP-GRASP-1 and myc-syntaxins were immunoprecipitated with anti-GFP antibody and analyzed by Western blot. (B) Lysates of COS-7 cells cotransfected with GFP-syntaxin 13 and full-length myc-GRASP-1 (1–837) or truncated myc-GRASP-1 constructs (1–695 or 695–837) were immunoprecipitated with anti-GFP antibody and analyzed by Western blot. Asterisk indicates background band. Arrows point to co-precipitated GRASP-1 proteins. (C) Binding assay using lysates of COS-7 cells expressing myc-syntaxin 13 with or without GFP-GRASP-1 and GMP-PNP-charged GST-rab4. Note that myc-syntaxin 13 is only isolated on the beads in the presence of GRASP-1. (D) Binding assay using lysate of COS-7 cells transfected with GFP-GRASP-1(594–837) and GST-syntaxins without transmembrane domain (ΔTM). GRASP-1 was analyzed by Western blot with antibody against GFP. (E) Binding assay of 35 S-labeled GRASP-1 and immobilized GST-syntaxin 13ΔTM.

Article Snippet: The following antibodies were obtained from commercial sources: rabbit anti-GRASP-1 (AB96361), mouse anti-β-actin, mouse anti-GluR2 (Chemicon), mouse anti-Rab4, mouse anti-EEA1 (BD Biosciences), mouse anti-FLAG, mouse anti-MAP2, mouse anti-αtubulin (Sigma), mouse anti-GFP (Roche), mouse anti-bassoon (Stressgen), rabbit anti-β-galactosidase (MP Biomedicals), mouse anti-β-galactosidase (Promega), rabbit anti-GluR1 (Calbiochem), mouse anti-HA (Roche), rabbit anti-myc (Upstate Biotechnology), mouse LAMP-1 (Stressgen), mouse anti-myc, rabbit anti-Rab5 (Santa Cruz Biotechnology), rabbit anti-syntaxin 13 (Synaptic Systems), human anti-EEA1, and mouse anti-human TfR (ATCC).

Techniques: Immunoprecipitation, Western Blot, Construct, Binding Assay, Expressing, Isolation, Transfection, Labeling

Journal: PLoS Biology

Article Title: Neuron Specific Rab4 Effector GRASP-1 Coordinates Membrane Specialization and Maturation of Recycling Endosomes

doi: 10.1371/journal.pbio.1000283

Figure Lengend Snippet: (A) Representative image of hippocampal neuron triple transfected at DIV13 for 4 d with GFP-Rab4, HA-GRASP-1, and myc-syntaxin 13 and labeled with anti-HA (blue) or anti-myc (red) antibodies. Magnified region of the cell body is shown to indicate the strong colocalization of GRASP-1, Rab4, and syntaxin 13. (B) Representative images of dendrites of hippocampal neurons transfected at DIV13 with GFP-GRASP-1 for 4 d and labeled with anti-syntaxin 13 (red). (C) Representative images of dendrites of hippocampal neurons transfected at DIV13 with GFP-GRASP-1 for 4 d and labeled with anti-Neep21 (red). (D) Representative images of dendrites of hippocampal neurons cotransfected at DIV13 for 4 d with myc-syntaxin 13 and control vector or HA-GRASP-1 and labeled with anti-myc (green), anti-HA (blue), and anti-Neep21 (red). (E) Representative images of dendrites of hippocampal neurons cotransfected at DIV13 for 4 d with GFP-Rab4, HA-Rab11, and control vector or myc-syntaxin 13ΔTM and labeled with anti-myc (blue) and anti-HA (red). (F) Percentage of colocalization between HA-GRASP-1 and myc-syntaxin 1 or myc-syntaxin 13 in neurons. (G) Percentage of colocalization between myc-syntaxin 13 and Neep21 in dendrites as indicated in (D). (H) Percentage of colocalization between GFP-Rab4 and HA-Rab11 domains in dendrites expressing myc-syntaxin 13ΔTM as indicated in (E). Error bars indicate S.E.M. ** p <0.005. *** p <0.0005. Bar in A is 10 µm; Bar in (B–E) is 1 µm.

Article Snippet: The following antibodies were obtained from commercial sources: rabbit anti-GRASP-1 (AB96361), mouse anti-β-actin, mouse anti-GluR2 (Chemicon), mouse anti-Rab4, mouse anti-EEA1 (BD Biosciences), mouse anti-FLAG, mouse anti-MAP2, mouse anti-αtubulin (Sigma), mouse anti-GFP (Roche), mouse anti-bassoon (Stressgen), rabbit anti-β-galactosidase (MP Biomedicals), mouse anti-β-galactosidase (Promega), rabbit anti-GluR1 (Calbiochem), mouse anti-HA (Roche), rabbit anti-myc (Upstate Biotechnology), mouse LAMP-1 (Stressgen), mouse anti-myc, rabbit anti-Rab5 (Santa Cruz Biotechnology), rabbit anti-syntaxin 13 (Synaptic Systems), human anti-EEA1, and mouse anti-human TfR (ATCC).

Techniques: Transfection, Labeling, Plasmid Preparation, Expressing

Journal: PLoS Biology

Article Title: Neuron Specific Rab4 Effector GRASP-1 Coordinates Membrane Specialization and Maturation of Recycling Endosomes

doi: 10.1371/journal.pbio.1000283

Figure Lengend Snippet: Endosomes can be viewed as mosaic distribution of Rab4, Rab5, and Rab11 domains that dynamically interact via effector proteins and SNAREs. The Rab5 domain allows entry into the early/sorting endosome, whereas the Rab4 and Rab11 domains contain the machinery that is necessary for sorting and recycling membranes and receptors back to the plasma membrane. (A) GRASP-1 binds to Rab4 and syntaxin 13 and couples Rab4 and Rab11 recycling endosomes. The complex formed between GRASP-1 and t-SNARE syntaxin 13 might mediate fusion between Rab4 and Rab11 endosomes. (B) Absence of GRASP-1 interferes with complex formation at the recycling step, causing cargo accumulation in early endosomes, impairment of receptor expression, and changes in spine morphology. (C) Overexpression of GRASP-1 leads to recruitment of syntaxin 13 and strongly couples Rab4 and Rab11 domains, causing accumulation of internalized receptors in recycling endosomes. Consistent with the observed decrease in AMPAR clusters , Caspase-3 cleavage of GRASP-1 might separate the N-terminal Rab4 domain from the C-terminal syntaxin 13 binding site and disrupt the coupling between Rab4 and Rab11 domains.

Article Snippet: The following antibodies were obtained from commercial sources: rabbit anti-GRASP-1 (AB96361), mouse anti-β-actin, mouse anti-GluR2 (Chemicon), mouse anti-Rab4, mouse anti-EEA1 (BD Biosciences), mouse anti-FLAG, mouse anti-MAP2, mouse anti-αtubulin (Sigma), mouse anti-GFP (Roche), mouse anti-bassoon (Stressgen), rabbit anti-β-galactosidase (MP Biomedicals), mouse anti-β-galactosidase (Promega), rabbit anti-GluR1 (Calbiochem), mouse anti-HA (Roche), rabbit anti-myc (Upstate Biotechnology), mouse LAMP-1 (Stressgen), mouse anti-myc, rabbit anti-Rab5 (Santa Cruz Biotechnology), rabbit anti-syntaxin 13 (Synaptic Systems), human anti-EEA1, and mouse anti-human TfR (ATCC).

Techniques: Expressing, Over Expression, Binding Assay

Journal: Traffic (Copenhagen, Denmark)

Article Title: Acute GARP Depletion Disrupts Vesicle Transport, Leading to Severe Defects in Sorting, Secretion and O ‐Glycosylation

doi: 10.1111/tra.70003

Figure Lengend Snippet: List of primary and secondary antibodies.

Article Snippet: STX5 , Santa Cruz, #sc‐365124 , Mouse , — , 1:100.

Techniques: Transduction

Journal: Traffic (Copenhagen, Denmark)

Article Title: Acute GARP Depletion Disrupts Vesicle Transport, Leading to Severe Defects in Sorting, Secretion and O ‐Glycosylation

doi: 10.1111/tra.70003

Figure Lengend Snippet: v‐SNARE GS15 is mislocalized in VPS54‐depleted cells. (A) RPE1 VPS54‐mAID cells were treated with AA as indicated, and cell lysates were probed with (top panel) anti‐GS15 (A) and anti‐GS28 (D). β‐Actin was used as a loading control. The bottom panels on (A) and (D) are the quantification of the blots from three independent experiments. (B) Airyscan microscopy of RPE1 VPS54‐mAID cells untreated or treated with AA for 3 h and co‐stained for GS15 and P230. (C) Colocalization analysis of GS15 and P230 of ≥ 30 cells was done by calculating Pearson's correlation coefficient. Statistical significance was calculated using a paired t ‐test. ** p ≤ 0.01. (F) WB analysis of RPE1 VPS54‐mAID cells treated with AA and probed with antibodies to STX5, STX6, STX10, VAMP4 and VTI1A, respectively. β‐Actin was used as a loading control.

Article Snippet: STX5 , Santa Cruz, #sc‐365124 , Mouse , — , 1:100.

Techniques: Control, Microscopy, Staining

Journal: Traffic (Copenhagen, Denmark)

Article Title: Acute GARP Depletion Disrupts Vesicle Transport, Leading to Severe Defects in Sorting, Secretion and O ‐Glycosylation

doi: 10.1111/tra.70003

Figure Lengend Snippet: Rapid VPS54 depletion results in the accumulation of GARP‐dependent vesicles and alteration of TGN morphology. (A) Transmission Electron Microscopy of high‐pressure frozen RPE1 VPS54‐mAID cells grown on sapphire discs before and after 3 h of AA treatment. “G” indicates Golgi stacks. Arrowheads point to vesicle‐like structures. Arrows indicate the enlarged vacuolar structures accumulated near the Golgi. Asterisks indicate the autophagosomes. Scale bar, 500 nm. (B) The graph represents the quantification of the total number of vesicles around the Golgi before and after 3 h of AA treatment. (C) Schematic of the cellular fractionation experiment to prepare P30 (Golgi), and P100 (Vesicle) fractions from control and 3 h AA treated groups. (D) WB analysis of TGN localized proteins (TGN46, CI‐MPR and CD‐MPR) in Golgi and vesicle fractions. (E) WB analysis of Golgi enzymes (B4GALT1, MGAT1, C1GALT1, GALNT2 and CPD) in Golgi and vesicle fractions. (F) WB analysis of SNAREs (STX5, GS15, STX10, STX6) in Golgi and vesicle fractions.

Article Snippet: STX5 , Santa Cruz, #sc‐365124 , Mouse , — , 1:100.

Techniques: Transmission Assay, Electron Microscopy, Cell Fractionation, Control