Journal: PLoS ONE

Article Title: Plasma membrane insertion of KCa2.3 (SK3) is dependent upon the SNARE proteins, syntaxin-4 and SNAP23

doi: 10.1371/journal.pone.0196717

Figure Lengend Snippet: (A) Co-localization of KCa2.3 with SNAP23 in hCMEC/D3 endothelial cells membrane footprints. hCMEC/D3 cells were transduced with BLAP-KCa2.3 Adenovirus. KCa2.3 was labeled with streptavidin-Alexa555 (Red, middle panel) and endogenous SNAP23 was labeled with α-SNAP23 Ab (Green, left panel). Co-localization is shown in yellow (Merge, Right panel). (B) Tracing analysis showing KCa2.3 (red) exhibits colocalization with SNAP-23 (green). (C) HEK cells were transfected with myc-tagged KCa2.3. KCa2.3 was immunoprecipitated followed by blotting for endogenous SNAP23. (D and E) hCMEC/D3 cells were transduced with BLAP-KCa2.3 Adenovirus. In (D) endogenous SNAP23 was immunoprecipitated followed by blotting for KCa2.3. In (E) , plasma membrane (PM) localized KCa2.3 was immunoprecipitated using α-streptavidin Ab followed by blotting for endogenous SNAP23. (F) Co-localization of KCa2.3 with Syntaxin-4 (Syn4) in hCMEC/D3 endothelial cell membrane footprints, as above. KCa2.3 was labeled with streptavidin-Alexa555 (Red, middle panel) and endogenous Syn4 was labeled with α-Syn4 Ab (Green, left panel). Co-localization is shown in yellow (Merge, Right panel). (G) Tracing analysis showing KCa2.3 (red) exhibits colocalization with Syn4 (green). (H and I) HEK cells were transfected with myc-tagged KCa2.3 and GFP-tagged Syn4. In (H) , KCa2.3 was immunoprecipitated followed by blotting for Syn4. In (I) , Syn4 was immunoprecipitated followed by blotting for KCa2.3. (J and K) hCMEC/D3 cells were transduced with BLAP-KCa2.3 Adenovirus. In (J) , endogenous Syn4 was immunoprecipitated followed by blotting for KCa2.3. In (K) , plasma membrane (PM) localized KCa2.3 was immunoprecipitated using α-streptavidin Ab followed by blotting for endogenous Syn4. In all blots, the lysate is shown in lane 1, the IgG control is shown in lane 2 and the IP is shown in lane 3. Scale bar = 10 μm in A and F.

Article Snippet: α-KCa2.3 Ab was obtained from Chemicon (Temecula, CA), α-myc Ab (9E10) was obtained from Covance (Richmond, CA), α-GFP Ab was obtained from Santa Cruz Biotechnology (Santa Cruz, CA), monoclonal α-streptavidin and α-SNAP-23 (AB4114) antibodies were obtained from Abcam (Cambridge, MA) and mouse α-syntaxin-4 was obtained from BD Transduction laboratories (San Jose, CA).

Techniques: Transduction, Labeling, Transfection, Immunoprecipitation

Journal: The Journal of Neuroscience

Article Title: Brain-Derived Neurotrophic Factor Promotes the Maturation of GABAergic Mechanisms in Cultured Hippocampal Neurons

doi: 10.1523/JNEUROSCI.22-17-07580.2002

Figure Lengend Snippet: BDNF upregulates GABAA receptor (GABAaR) protein and presynaptic proteins. Cells were cultured in the presence or absence of 50 ng/ml BDNF for 7–10 d, and the extracts were analyzed by Western blot for NR1, GluR1, and GABAAR β2/3 (A), and syntaxin, synaptobrevin, and synaptophysin (B). Immunoreactive signals were normalized to the control. Insets are representative Western blots. A blot for β-actin for normalization is also shown. Data are means ± SEM of three independent experiments. *p < 0.05 versus control, Student's t test.

Article Snippet: The membrane was incubated in PBS containing 0.05% Tween 20 and 5% nonfat dry milk for 1 hr and then exposed to a primary antibody against synaptophysin (1:200, mouse; Boehringer Mannheim Biochemica), synaptobrevin (1:500, rabbit; Wako, Osaka, Japan), syntaxin (1:300, mouse; Wako), GluR1 (1:300, rabbit;Chemicon), NR1 (1:300, rabbit; Chemicon), or GABA A Rβ2/3 (only the β3 subunit was detected in our culture; 1:300, mouse; Upstate Biotechnology, New York, NY) in 0.05% Tween 20–PBS at 4°C overnight and then to horseradish peroxidase–conjugated secondary antibody for appropriate IgG.

Techniques: Cell Culture, Western Blot

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: 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

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: