Journal: Journal of cell science

Article Title: Patch repair protects cells from the small pore-forming toxin aerolysin.

doi: 10.1242/jcs.261018

Figure Lengend Snippet: Fig. 6. Annexins minimally resist aerolysin with limited shedding. HeLa cells were transfected with control (CTL), A1, A2 or A6 siRNA for 3 days and then (A,B) lysed for western blot analysis, or (C) challenged with 31–2000 HU/ml aerolysin (Aero), SLO or ILY for 30 min at 37°C. PI uptake was analyzed by flow cytometry. (A) Portions of the blot were probed with antibodies against A1, A2, A6 or β-actin followed by HRP-conjugated secondary antibodies. (B) Quantification of the knockdown efficiency compared to control siRNA is shown. (D,E) HeLa cells were transfected with A6–YFP and challenged with sublytic toxin concentrations (62 HU/ml pro-aerolysin or aerolysin, or 250 HU/ml SLO) in imaging buffer. The cells were imaged by confocal microscopy for ∼45 min at 37°C and then lysed with 1% Triton X-100. The time to half maximal (t1/2) (D) cytosolic depletion or (E) membrane accumulation of A6 is shown. (F) Shedding of annexin A6+ microvesicles was manually counted and expressed as microvesicles shed/cell/min. (G) HeLa cells were challenged for 15 min at 37°C with sublytic doses of aerolysin, SLO, mass equivalent of AeroY221G or sufficient trypsin to activate pro-aerolysin. Cells were pelleted at 2000 g for 5 min to yield cell pellets (denoted C). Cell supernatants were spun at 100,000 g for 40 min at 4°C and the microvesicle pellet (MV) collected. Fractions were analyzed by western blotting using anti-aerolysin, 6D11 anti- SLO, CPTC-A1-3 anti-annexin A1, anti-Alix, MANLAC-4A7 anti-lamin A/C or AC-15 anti-β-actin. Graphs show the mean (C,F) or geometric mean (D,E). Data points represent (C) individual experiments or (D,E) individual cells from three independent experiments. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001; ns, not significant (one-way ANOVA with Tukey post test). Asterisks in G indicates non-specific bands.

Article Snippet: Anti-SLO antibody 6D11 mAb (catalog: NBP1-05126) was from Novus Biologicals (Littleton, CO, USA).

Techniques: Transfection, Control, Western Blot, Flow Cytometry, Knockdown, Imaging, Confocal Microscopy, Membrane

Journal: eLife

Article Title: Syntaxin-6 delays prion protein fibril formation and prolongs the presence of toxic aggregation intermediates

doi: 10.7554/eLife.83320

Figure Lengend Snippet: ( A ) Non-infected PK1 cells and persistently infected PK1 cells (iS7) were immuno-stained with anti-PrP antibodies 6D11 and 5B2 (green), with anti-syntaxin-6 antibody (red) and with DAPI (blue). Förster resonance energy transfer (FRET) analysis reveals interaction in perinuclear compartments (wide arrows) and at membranes in infected cells (narrow arrows). Panels show zoomed regions of images in . ( B, C ) In vitro prion replication by protein misfolding cyclic amplification (PMCA) using Stx6 +/+ and Stx6 -/- mouse brains as substrate. PMCA reactions were seeded with RML prions from terminally ill mice and subjected to PMCA for 96 cycles over 48 hr. ( B ) Representative western blot (ICSM35) after PK digestion. Molecular weight markers are indicated on the left. ( C ) The PrP Sc signal was quantified using densitometry and normalized to the control unamplified reaction. Bar graphs each represent mean ± SD of biological replicates from three separate mice, each blotted as two technical replicates. Source data is available at https://doi.org/10.17632/yggpkrgnx8.1 .

Article Snippet: Following five washes, the cells were incubated with anti-syntaxin-6 (Cell Signaling Technologies [#2869], clone C34B2, 1:300) and/or anti-PrP (BioLegend [808001] clone 6D11, 1:10,000; Santa Cruz [sc-47730], 5B2, 1:500; Sigma [P0110], clone 8H4, 1:500) in sterile-filtered 0.25× SuperBlock in PBS overnight at 4°C, followed by secondary antibodies (AlexaFluor 647-AffiniPure Goat Anti-Rabbit IgG (H+L) and/or Rhodamine Red-X (RRX) AffiniPure Goat Anti-Mouse IgG (H+L), 1:1000) and a DNA counterstain (DAPI; 1:10,000) in 0.25× SuperBlock overnight at 4°C.

Techniques: Infection, Staining, Förster Resonance Energy Transfer, In Vitro, Protein Misfolding Cyclic Amplification, Western Blot, Molecular Weight, Control

Journal: eLife

Article Title: Syntaxin-6 delays prion protein fibril formation and prolongs the presence of toxic aggregation intermediates

doi: 10.7554/eLife.83320

Figure Lengend Snippet: ( A, B ) PixFRET analysis of non-infected PK1 cells and persistently infected PK1 cells (iS7). Cells were immuno-stained with anti-PrP antibodies 6D11, 8H4, or 5B2 (green), with anti-syntaxin-6 antibody (STX6, red) and with DAPI. ( C ) Schematic of binding locations of antibodies used in FRET analysis (5B2, 6D11, 8H4). Numbers represent putative epitopes. ( C ) was adapted from Figure 2B from .

Article Snippet: Following five washes, the cells were incubated with anti-syntaxin-6 (Cell Signaling Technologies [#2869], clone C34B2, 1:300) and/or anti-PrP (BioLegend [808001] clone 6D11, 1:10,000; Santa Cruz [sc-47730], 5B2, 1:500; Sigma [P0110], clone 8H4, 1:500) in sterile-filtered 0.25× SuperBlock in PBS overnight at 4°C, followed by secondary antibodies (AlexaFluor 647-AffiniPure Goat Anti-Rabbit IgG (H+L) and/or Rhodamine Red-X (RRX) AffiniPure Goat Anti-Mouse IgG (H+L), 1:1000) and a DNA counterstain (DAPI; 1:10,000) in 0.25× SuperBlock overnight at 4°C.

Techniques: Infection, Staining, Binding Assay

Journal: eLife

Article Title: Syntaxin-6 delays prion protein fibril formation and prolongs the presence of toxic aggregation intermediates

doi: 10.7554/eLife.83320

Figure Lengend Snippet: PixFRET analysis of control cells. iS7 cells were immuno-stained with anti-PrP antibodies 6D11, 8H4, or 5B2 (green) only, with anti-syntaxin-6 antibody (STX6, red) only, or with secondary antibodies only and with DAPI.

Article Snippet: Following five washes, the cells were incubated with anti-syntaxin-6 (Cell Signaling Technologies [#2869], clone C34B2, 1:300) and/or anti-PrP (BioLegend [808001] clone 6D11, 1:10,000; Santa Cruz [sc-47730], 5B2, 1:500; Sigma [P0110], clone 8H4, 1:500) in sterile-filtered 0.25× SuperBlock in PBS overnight at 4°C, followed by secondary antibodies (AlexaFluor 647-AffiniPure Goat Anti-Rabbit IgG (H+L) and/or Rhodamine Red-X (RRX) AffiniPure Goat Anti-Mouse IgG (H+L), 1:1000) and a DNA counterstain (DAPI; 1:10,000) in 0.25× SuperBlock overnight at 4°C.

Techniques: Control, Staining

Journal: bioRxiv

Article Title: Large Extracellular Vesicles Induce Stronger Prion Infection in Cell Culture than Small Extracellular Vesicles

doi: 10.1101/2023.03.01.530593

Figure Lengend Snippet: (a-d) CAD5-RML (a, b) or N2a-PK1-RML (c, d) lEVs, sEVs, and whole-cell lysate were treated with proteinase K (PK+, a, c), and PrPres was detected using a mix of prion antibodies 6D11 and AH6. Untreated (PK-) samples (b, d) demonstrated the presence of total PrP (PrP C + PrP Sc ). For visualization purposes, all images were enhanced with brightness and contrast. (e,f) Comparison of the relative densities of PrPres bands in lEVs, sEVs, and whole-cell lysate normalized to PrP PK-signal in whole-cell lysate from CAD5-RML (e) or N2a-PK1-RML (f) cells. Bars represent the mean with SEM of biological triplicates, each with technical triplicates (3 different protein loadings on gel – 6, 3, or 1.5 μg per line).

Article Snippet: The membranes were blocked using Superblock™ solution (Thermo Fisher Scientific) for 1 h. After blocking, the membranes were incubated with 6D11 primary antibody diluted in Superblock™ solution at 4 °C overnight.

Techniques: