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: Journal of Virology

Article Title: Glycosylated NS3/NS3A protein of bluetongue virus facilitates efficient viral egress via lipid raft anchoring

doi: 10.1128/jvi.02144-25

Figure Lengend Snippet: N-linked glycosylation drives plasma membrane accumulation of NS3/NS3A. ( A ) The stability of NS3/NS3A WT and NS3/NS3A N150Q proteins was examined in both transfected (top panels) and BTV-20-infected cells (bottom panels; MOI = 10). For transfection assays, HEK-293T cells were transfected with plasmids expressing NS3/NS3A WT or the N150Q mutant and treated with cycloheximide (CHX; 100 μg/mL) at 18 h post-transfection (designated as 0 h post-CHX treatment) to block de novo protein synthesis. For infection assays, MDOK cells were infected with BTV-20 WT or BTV-20 N150Q and treated with CHX (100 μg/mL) at 10 h post-infection (designated as 0 h post-CHX treatment). Cells were harvested at the indicated time points and analyzed by Western blotting. ( B ) Quantification of NS3/NS3A protein levels shown in panel A was performed by ImageJ densitometric analysis. Protein levels at each time point were normalized to the corresponding 0 h post-CHX treatment (18 h post-transfection or 10 h post-infection, respectively). ( C ) Subcellular localization of NS3/NS3A in MDOK cells infected with BTV-20 WT or BTV-20 N150Q (MOI = 5, 12 h.p.i.). NS3/NS3A (red) was co-stained with ER marker anti-calnexin (green) or Golgi marker anti-syntaxin 6 (green). Fluorescence distribution was evaluated using line-scan intensity profiles. Scale bar, 5 µm. ( D ) Subcellular localization of NS3/NS3A in MDOK cells infected with BTV-20 WT or BTV-20 N150Q (MOI = 5, 12 h.p.i.). NS3/NS3A (red) was co-stained with plasma membrane marker WGA-Alexa Fluor 488 (green). Line-scan intensity profiles are shown. Scale bar, 5 µm. ( E ) Plasma membrane isolation of HEK-293T cells transfected with NS3/NS3A WT or N150Q mutant, followed by Western blot analysis. PM (plasma membrane fraction); NPM (non-plasma membrane fraction); Total (plasma membrane fraction + non-plasma membrane fraction). ( F ) Quantification of NS3/NS3A at the plasma membrane fraction was analyzed by Image J from panel E (* P < 0.05, two-tailed unpaired t-test). ( G ) Confocal imaging of HeLa cells co-transfected with NS3/NS3A (WT or N150Q, red) and VP2 (green) or VP5 (green), showing their subcellular colocalization. Colocalization was assessed by line-scan intensity profiles.

Article Snippet: Commercial antibodies used in this study included anti-FLAG (DYKDDDDK) monoclonal antibody (1:1,000 for immunofluorescence assay [IFA], 1:10,000 for western blotting [WB]; 66008-4-Ig, Proteintech), anti-HA polyclonal antibody (1:100 for IFA, 1:1,000 for WB; 51064-2-AP, Proteintech), anti-β-actin monoclonal antibody (1:10,000 for WB; 66009-1-Ig, Proteintech), anti-Calnexin polyclonal antibody (1:200 for IFA; 10427-2-AP, Proteintech), anti-Syntaxin 6 polyclonal antibody (1:200 for IFA; 10841-1-AP, Proteintech), anti-Flotillin 1 monoclonal antibody (1:100 for IFA; 67968-1-Ig, Proteintech), and anti-Filamin A (FLNA) monoclonal antibody (1:1,000 for WB; 67133-1-Ig, Proteintech).

Techniques: Glycoproteomics, Clinical Proteomics, Membrane, Transfection, Infection, Expressing, Mutagenesis, Blocking Assay, Western Blot, Staining, Marker, Fluorescence, Isolation, Two Tailed Test, Imaging

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: