Journal: BMC Biology

Article Title: The eggshell is required for meiotic fidelity, polar-body extrusion and polarization of the C. elegans embryo

doi: 10.1186/1741-7007-4-35

Figure Lengend Snippet: GNA-2 deficiency results in a polarity-osmotic defective (Pod) phenotype . (A) Wild type or gna-2(qa705) stained for DNA (Hoechst stain; blue) and PAR-3 (pink), PGL-1 (pink) or PIE-1 (green), or wild type expressing PAR-2::GFP fed OP50 (control) or gna-2 RNAi and stained for DNA (Hoechst; blue) and GFP (green) (second row). Embryos are at the one-cell stage except wild type stained for PIE-1, which is at the two-cell stage. Wild-type embryos are oriented with anterior (maternal DNA, polar-body end) on the left. gna-2(qa705) or gna-2(RNAi) does not extrude polar bodies therefore it was not possible to determine the maternal end. Images are overlays of Hoechst and antibody staining. (B) DIC images showing osmotic sensitivity of wild type or gna-2(qa705) dissected in water, 150 mM KCl or 300 mM KCl. Arrows point to the eggshell. (C) Plasma-membrane staining of gna-2(qa705) with the lipophilic dye, FM 4–64 (red). In wild type, only the first polar body stains (white arrow). DIC images are shown on the left.

Article Snippet: Lipophilic dye permeability was tested by DIC/UV microscopy of living embryos dissected in 150 mM KCl containing 30 μM FM 4–64 (Molecular Probes).

Techniques: Staining, Expressing

Journal: PLoS Neglected Tropical Diseases

Article Title: NLRP3 Inflammasome Activation by Paracoccidioides brasiliensis

doi: 10.1371/journal.pntd.0002595

Figure Lengend Snippet: NLRP3 inflammasome activation by P. brasiliensis requires K + efflux, ROS production, lysosomal acidification and cathepsin B release. BMDCs were pretreated for 1( Methods section) of (A, B) glibenclamide and KCL, (C, D) NAC and APDC, or (E, F) bafilomycin and CA-074. Then, the cells were infected with P. brasiliensis (Pb) at an MOI of 1 for 24 h. The supernatants from the cultures were harvested for IL-1β and TNF-α assays using ELISA. The data are expressed as the mean ± the SD of two to three independent experiments conducted in triplicates. * p ≤0.05.

Article Snippet: In some experiments, the phagocytic cells were pre-treated (for 1 h) with either the NF-κB inhibitors Bay11-7082 (5 µM) and celastrol (5 µM) (both from InvivoGen), the cathepsin B inhibitor CA-074Me (50 µM) (Sigma-Aldrich), the endosomal acidification inhibitor bafilomycin (250 nM) (InvivoGen), the ATP-sensitive K+ channel inhibitor glibenclamide (150 µM) (InvivoGen), KCL (100 mM) (Sigma-Aldrich), ROS scavengers/inhibitors NAC (N-acetyl-L-cysteine) (20 mM) or APDC (2R,4R)-4 aminopyrrolidine-2,4-dicarboxylic acid) (100 µM) (both from Sigma-Aldrich), the caspase-1 inhibitor AC-Y-VAD-CHO (50 µM) (Santa Cruz Biotechnology), the caspase-8 inhibitor Z-IETD-FMK (50 µM) (R & D Systems), the Syk inhibitor piceatannol (3,4′,3′,5-trans-trihydroxystilbene) (25 µM) (InvivoGen) or the Myd88 inhibitory peptide Pepinh-MYD (50 µM) (InvivoGen).

Techniques: Activation Assay, Infection, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Biological Chemistry

Article Title: Parkinson Disease-linked Vps35 R524W Mutation Impairs the Endosomal Association of Retromer and Induces α-Synuclein Aggregation *

doi: 10.1074/jbc.M115.703157

Figure Lengend Snippet: Parkinson disease-linked Vps35 point mutations increase the production of α-synuclein-positive aggregates. A , SH-SY5Y cells were untreated or were induced by KCl treatment to form α-synuclein aggregates ( arrows ), and immunofluorescence

Article Snippet: SH-SY5Y cells grown on glass coverslips were incubated with 50 m m KCl (Sigma-Aldrich) for 60 min at 37 °C.

Techniques: Immunofluorescence

Journal: Nature Communications

Article Title: Atomically resolved three-dimensional structures of electrolyte aqueous solutions near a solid surface

doi: 10.1038/ncomms12164

Figure Lengend Snippet: Three-dimensional images of mica-electrolyte solution interfaces. ( a ) 3D AFM image of a KCl (aq.) solution (0.2 M KCl). The image shows a monolayer of adsorbed K + ions (light red) topped by two hydration layers (lighter stripes). The hydration layers (∼0.3 nm thick) follow the atomic corrugation of the surface. ( b ) 3D AFM image of a mica-KCl (aq.) interface (∼4 M). The interface is divided in two main regions, an ordered liquid layer extending up to 2 nm from the mica and the bulk solution above it. ( c ) 3D AFM image of a mica-NaCl (aq.) interface. Atomic scale order is seen in xy, xz and yz planes. These 3D maps show the variations of the phase shift of the tip's oscillation. Supplementary Movies 1 (52 s), 2 (105 s) and 3 (105 s) include the complete 3D data, respectively, of a – c . The 3D AFM experiments were performed at ∼300 K.

Article Snippet: NaCl and KCl solutions were prepared from 99.5% (NaCl) and 99.0% (KCl) salts (Sigma-Aldrich) dissolved in ultrapure water (18.2 MΩ, pH=5.5).

Techniques:

Journal: Molecular & Cellular Proteomics : MCP

Article Title: An Inducible Retroviral Expression System for Tandem Affinity Purification Mass-Spectrometry-Based Proteomics Identifies Mixed Lineage Kinase Domain-like Protein (MLKL) as an Heat Shock Protein 90 (HSP90) Client *

doi: 10.1074/mcp.O115.055350

Figure Lengend Snippet: pRSHIC allows inducible, dose-dependent, and reversible expression of SH-tagged bait proteins. ( A–F ) Flow cytometry and immunoblot analysis of K-562 RIEP ( A, D ), HT-29 RIEP ( B, E ) and KCL-22 RIEP ( C, F ) GFP cells, untreated or treated with 1–2

Article Snippet: HEK293T was obtained from ATCC (Manassas, VA) and K-562 and KCL-22 from DSMZ (Braunschweig, Germany).

Techniques: Expressing, Flow Cytometry, Cytometry

Journal: Biophysical Journal

Article Title: Kinetics of K+ Occlusion by the Phosphoenzyme of the Na+,K+-ATPase

doi: 10.1016/j.bpj.2010.11.038

Figure Lengend Snippet: Sequential mixing stopped-flow fluorescence transients of Na + ,K + -ATPase from pig kidney and shark rectal gland noncovalently labeled with RH421. Na + ,K + -ATPase (60 μ g/mL) was premixed with a small volume of 0.5 M Na 2 ATP to a final concentration of 4 mM ATP. A few seconds later, the enzyme was mixed with an equal volume of a KCl solution (0.6 mM after mixing). The final Na + ,K + -ATPase concentration was 30 μ g/mL, and the RH421 concentration was 400 nM. Both the enzyme suspensions and the KCl solutions were in a buffer containing 30 mM imidazole, 40 mM NaCl, 5 mM MgCl 2 , and 1 mM EDTA (pH 7.4, 24°C). The total ionic strength was maintained at a constant value by the addition of choline chloride to the enzyme suspension and the KCl solutions ([choline chloride] + [KCl] = 90 mM). The fluorescence of membrane-bound RH421 was measured at an excitation wavelength of 577 nm at emission wavelengths of ≥665 nm (RG665 glass cutoff filter). The calculated observed rate constants were 67 (±2) s −1 for the pig enzyme and 120 (±11) s −1 (82% of the total amplitude) and 16 (±3) s −1 (18%) for the shark enzyme.

Article Snippet: The origins of the various reagents used were: imidazole (≥99%; Sigma, Castle Hill, Australia), NaCl (suprapure; Merck, Kilsyth, Australia), KCl (analytical grade; Merck), choline chloride (99%; Sigma), MgCl2 ·6H2 O (analytical grade; Merck), EDTA (99%; Sigma), ATP disodium·3H2 O (special quality; Roche, Castle Hill, Australia), NaOH (analytical grade; Merck), and HCl (0.1 N Titrisol solution; Merck).

Techniques: Flow Cytometry, Fluorescence, Labeling, Concentration Assay

Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

Article Title: Synaptic mechanisms of temporal diversity in the lateral geniculate nucleus of the thalamus

doi: 10.1523/JNEUROSCI.4046-12.2013

Figure Lengend Snippet: Early synaptic inhibition is mediated by chloride. Recording from a X L cell with 3M KCl pipette solution. A) Three minutes after impalement, the leading inhibition to a same-sign contrast center stimulus was evident as shown by PSTH (top), the spike raster

Article Snippet: We further investigated the ionic basis of the early inhibition at a cellular level by recording from 4 XL cells with KCl (3M) in the pipette rather than the control KAc solution.

Techniques: Inhibition, Transferring