Journal: The Journal of Neuroscience

Article Title: Psychosine Accumulates in Membrane Microdomains in the Brain of Krabbe Patients, Disrupting the Raft Architecture

doi: 10.1523/JNEUROSCI.5597-08.2009

Figure Lengend Snippet: Redistribution of raft marker proteins accompanies psychosine accumulation in TWI lipid rafts. Lipid raft fractions from wild-type (W) and twitcher (T) nervous tissue were subjected to Western blot analysis. Representative blots are presented and show distributions of both raft Flotillin-2 (Flot 2)- and Caveolin-1 (Cav 1)- and nonraft (P115)-markers. These blots show that raft-marker distribution in the twitcher at all time points and in all tissues is disrupted in conjunction with the accumulation of psychosine. Proteins were also analyzed in total homogenates (To) and pellets (Pe). Actin was used as a house-keeping gene in Pe and To fractions.

Article Snippet: Antibodies used in this study were the following: anti-flotillin-2, P115 (Beckton Dickson), caveolin-1 (Cell Signaling), PKC (Santa Cruz), phospho-PKC (Cell Signaling), and actin (Millipore Bioscience Research Reagents).

Techniques: Marker, Western Blot

Journal: The Journal of Neuroscience

Article Title: Psychosine Accumulates in Membrane Microdomains in the Brain of Krabbe Patients, Disrupting the Raft Architecture

doi: 10.1523/JNEUROSCI.5597-08.2009

Figure Lengend Snippet: Psychosine accumulation disrupts raft architecture in brains of human Krabbe patients. A, Mass spectrometric analysis of human samples is expressed in picomoles per gram and reveals that psychosine preferentially accumulates in lipid rafts in humans with Krabbe disease. B, Fluorometric cholesterol assays of human samples are expressed as a percentage of the total and show that cholesterol accumulation accompanies the accumulation of psychosine. C, Western blotting was used to analyze fractions prepared from human tissue for distributions of the lipid raft-marker proteins Flotillin-2 (Flot 2) and Caveolin-1 (Cav-1). The results show a disruption of these proteins in a pattern similar to that observed in the TWI mouse. P115 was again used as a marker for nonrafts.

Article Snippet: Antibodies used in this study were the following: anti-flotillin-2, P115 (Beckton Dickson), caveolin-1 (Cell Signaling), PKC (Santa Cruz), phospho-PKC (Cell Signaling), and actin (Millipore Bioscience Research Reagents).

Techniques: Western Blot, Marker

Journal: Journal of Extracellular Vesicles

Article Title: Culture conditions greatly impact the levels of vesicular and extravesicular Ago2 and RNA in extracellular vesicle preparations

doi: 10.1002/jev2.12366

Figure Lengend Snippet: Ago2 is present in both vesicular and nonvesicular forms in EV‐depleted serum conditions . (a) Western blot analyses of DKs‐8 TCL, LEV, and DG fractions from iodixanol cushions from the different media conditions probed for Ago2, Flotillin‐1, and CD63. Middle panel : Relative intensities of the Ago2, Flotillin‐1, and CD63 bands in the cushion DG fractions from the different media conditions, normalized to the band intensity of the highest SEV lane (either Fraction 6 or 7) (n = 4 or 5). Bottom panel : Percentages of protein bands found associated with Fractions 5–8 and 9–12. Data plotted as Mean+/‐SE. (b) Western blot analyses of DLD‐1 TCL, LEV, and DG fractions from iodixanol cushions from the different media conditions probed for Ago2, Flotillin‐1, and CD63. Middle panel : Relative intensities of the Ago2, Flotillin‐1, and CD63 bands in the cushion DG fractions from the different media conditions, normalized to the band intensity of the highest SEV lane (either Fraction 6 or 7) (n = 4). Bottom panel : Percentages of protein bands found associated with Fractions 5–8 and 9–12. Data plotted as Mean+/‐SE. (c) Western blot analyses of DKs‐8 TCL, LEV, and DG fractions from 4 h UC to DG from EV‐depleted FBS in DMEM condition probed for Ago2, Flotillin‐1, and CD63. Middle panel : Relative intensities of the Ago2, Flotillin‐1, and CD63 bands in the DG fractions from 4 h UC to DG from EV‐depleted FBS in DMEM condition, normalized to the band intensity of the highest SEV lane (either Fraction 6 or 7) (n = 4). Bottom panel : Percentages of protein bands found associated with Fractions 5–8 and 9–12. Data plotted as Mean ± SE.

Article Snippet: Mouse anti‐Flotillin‐1 (cat no. 610820) was purchased from BD BioSciences.

Techniques: Western Blot

Journal: Journal of Extracellular Vesicles

Article Title: Culture conditions greatly impact the levels of vesicular and extravesicular Ago2 and RNA in extracellular vesicle preparations

doi: 10.1002/jev2.12366

Figure Lengend Snippet: Cell culture media components affect the abundance of Ago2, hnRNP A2/B1, and EV markers in EVs . (a) Representative traces from nanoparticle tracking analysis of DKs‐8 SEVs obtained from different conditioning media. (b) Quantitation of DKs‐8 particle numbers in our SEV preparations by nanoparticle tracking analysis (n = 4). Data were shown as Mean ± SE. ns, not significant. (c) Representative TEM images of DKs‐8 SEVs obtained from the different conditioning media. Scale bar shows 200 nm. (d) Western blot assessments of DKs‐8 total cell lysate and SEVs (loading based on equal protein concentration and equal vesicle number) from the different conditioning media, probing for Ago2, hnRNP A2/B1, Flotillin, and CD63 (n = 4 or 5). Relative intensities of the Ago2, hnRNP A2/B1, Flotillin and CD63 bands of SEVs in (c), normalized to the band intensities of serum‐free DMEM. Paired t test, * p < 0.05, ** p < 0.01, *** p < 0.001, ns, not significant. (e) Different concentrations of SEVs obtained from DKs‐8 cells conditioned with the different conditioning media were dotted on nitrocellulose membranes and probed with anti‐Ago2 (rat antibody) or anti‐CD63 antibodies in the presence (Tween) or absence (No Tween) of 0.1% Tween‐20. Right: Quantitation of Ago2 dots in presence or absence of Tween (n = 3).

Article Snippet: Mouse anti‐Flotillin‐1 (cat no. 610820) was purchased from BD BioSciences.

Techniques: Cell Culture, Quantitation Assay, Western Blot, Protein Concentration

Journal: Journal of Extracellular Vesicles

Article Title: Culture conditions greatly impact the levels of vesicular and extravesicular Ago2 and RNA in extracellular vesicle preparations

doi: 10.1002/jev2.12366

Figure Lengend Snippet: Hollow fiber bioreactors greatly affect EV populations and the distribution of RBPs on density gradients . (a) Western blot analyses of DKs‐8 TCL, LEV, and DG fractions from cushion density gradients for CDM‐HD in DMEM (serum‐free condition) and EV‐depleted FBS in DMEM probed for Ago2, hnRNP A2/B1, Flotillin‐1, and CD63. Middle panel: Relative intensities of the Ago2, hnRNP A2/B1, Flotillin‐1, and CD63 bands in the cushion density gradient fractions from the different media conditions, normalized to the band intensity of the highest SEV lane (either Fraction 6 or 7) (n = 8 and 6, respectively). Bottom panel: Percentages of Ago2, hnRNP A2/B1 (abbreviated as A2/B1), Flotillin‐1 (abbreviated as Flot‐1), and CD63 bands found associated with Fractions 5–7, 8–10, and 11–12. Data plotted as Mean ± SE. * p < 0.05, ** p < 0.01, *** p < 0.001. (b) Western blot analyses of DLD‐1 TCL, LEV, and DG fractions from cushion density gradients for the different media conditions probed for Ago2, hnRNP A2/B1, Flotillin‐1, and CD63. Middle panel: Relative intensities of the Ago2, hnRNP A2/B1, Flotillin‐1, and CD63 bands in the cushion density gradient fractions from the different media conditions, normalized to the band intensity of the highest SEV lane (either Fraction 6 or 7) (n = 5 and 4, respectively). Bottom panel: Percentages of Ago2, hnRNP A2/B1 (abbreviated as A2/B1), Flotillin‐1 (abbreviated as Flot‐1), and CD63 bands found associated with Fractions 5–7, 8–10, and 11–12. Data plotted as Mean ± SE. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Mouse anti‐Flotillin‐1 (cat no. 610820) was purchased from BD BioSciences.

Techniques: Western Blot

Journal: ASN NEURO

Article Title: SOD1 G93A Astrocyte-Derived Extracellular Vesicles Induce Motor Neuron Death by a miRNA-155-5p-Mediated Mechanism

doi: 10.1177/17590914231197527

Figure Lengend Snippet: Morphological characterization and size distribution of extracellular vesicles present in astrocyte CM. (a) TEM micrographs (negative staining) from extracellular vesicles isolated from astrocyte CM. Bars: 100 nm. (b) Frequency distribution of EV diameter as a percentage of the total number of vesicles from the three independent experiments. (c) Representative western blot of the exosomes from non-Tg or SOD1 G93A astrocyte CM using antibodies against TSG101, flotillin-1, and human SOD1.

Article Snippet: Primary antibodies include rabbit monoclonal anti-TSG101, 1:1,000 (Abcam, Cambridge, UK, Cat# ab125011, RRID: AB_10974262); mouse monoclonal anti-flotillin-1, 1:500 (BD Biosciences, Franklin Lakes, NJ, USA, Cat# 610820, RRID: AB_398139); rabbit polyclonal anti-human SOD1 antibody kindly provided by Prof. Mónica Marin from Facultad de Ciencias, UdelaR, 1:500 ( ); and rabbit polyclonal anti-BIP, 1:1,000 (Abcam, ab21685, RRID: AB_2119834).

Techniques: Negative Staining, Isolation, Western Blot