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:

Journal: Molecular Vision

Article Title: Age-dependent changes in rat lacrimal gland anti-oxidant and vesicular related protein expression profiles

doi:

Figure Lengend Snippet: Western blotting antibodies used to compare SNARE expression levels in aging and control rat LG.

Article Snippet: Syntaxin 1A , Santa Cruz SC 12736 , Mouse polyclonal , 35 kDa , 200 μg/ml.

Techniques: Western Blot, Expressing, Control, Molecular Weight, Concentration Assay

Journal: Molecular Vision

Article Title: Age-dependent changes in rat lacrimal gland anti-oxidant and vesicular related protein expression profiles

doi:

Figure Lengend Snippet: Effect of aging on the expression of the ratio of ( A ) Rab 3d/GAPDH, ( B ) Syntaxin 1A/GAPDH, and ( C ) Vamp 2/GAPDH in LG. Tissues from both groups were excised and homogenates were analyzed by western blot (*p<0.05, Mann–Whitney U test). Results are representative of three independent experiments.

Article Snippet: Syntaxin 1A , Santa Cruz SC 12736 , Mouse polyclonal , 35 kDa , 200 μg/ml.

Techniques: Expressing, Western Blot, MANN-WHITNEY