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

Journal: iScience

Article Title: SARS-CoV-2 antigen-carrying extracellular vesicles activate T cell responses in a human immunogenicity model

doi: 10.1016/j.isci.2023.108708

Figure Lengend Snippet: Characterization of 293F cell-derived EVs carrying SARS-CoV-2 spike protein (A) Schematic of EV engineering strategy using 293F cells including SARS-CoV-2 spike expression construct (see Figure S1 ) with spike protein depicted in blue. Schematic created with Biorender.com . (B) Histogram plot of particle size distribution comparing 293F EVs and Spike EVs as determined by NTA. The number of particles falling within each size bin was normalized to total particle counts and are presented as the proportion of total particles. Data represent the mean ± standard error of the mean (SEM). N = 3 independent experiments. Statistical comparisons were carried out by unpaired, two-tailed Student’s t test where p < 0.05 would be considered significant. (C) Mean and mode particle size of 293F EVs and Spike EVs as determined by NTA. Data represent the mean ± standard error of the mean (SEM). N = 3 independent experiments. Statistical comparisons were carried out by unpaired, two-tailed Student's t test where p < 0.05 would be considered significant. n.s. indicates not significant. (D) Multiplexed bead-based profiling assay of 37 surface antigens on 293F EVs and Spike EVs confirming the presence of expected EV surface markers. Data represent the median fluorescent intensity (MFI) of APC signal (reflecting CD63-APC, CD81-APC and CD9-APC counterstaining) following the subtraction of isotype controls. N = 1 independent experiment. (E) Representative western blots characterizing the presence of SARS-CoV-2 spike protein in Spike EVs and markers known to be present in EV preparations (CD63, CD9, Flotillin, TSG101, GAPDH) as well as the absence of cell contaminant markers (GM130, Calnexin). (F) Confirmation of SARS-CoV-2 spike protein presence in Spike EVs through competitive Spike-ACE2 binding assay. Spike EVs inhibit the binding of fluorescently labeled, recombinant SARS-CoV-2 spike protein competitively, in a dose-responsive manner, while 293F EVs demonstrate little inhibition. Data are mean ± SEM. N = 2 independent experiments and 2 technical replicates per sample per run. (G) Transmission electron micrographs of unstained Spike EVs confirming expected EV morphology. SARS-CoV-2 spike protein presence on 293F Spike EVs was also visually confirmed using immunogold labeling where 10 nm gold particles are seen associating with 293F Spike EVs.

Article Snippet: Mouse Anti-Flotillin-1, Clone 18 , BD Transduction Laboratories , Cat#610820; RRID: AB_398139.

Techniques: Derivative Assay, Expressing, Construct, Two Tailed Test, Western Blot, Binding Assay, Labeling, Recombinant, Inhibition, Transmission Assay

Journal: The Journal of Neuroscience

Article Title: Reggies/Flotillins Regulate Retinal Axon Regeneration in the Zebrafish Optic Nerve and Differentiation of Hippocampal and N2a Neurons

doi: 10.1523/JNEUROSCI.0870-09.2009

Figure Lengend Snippet: Reggie downregulation reduces axon outgrowth. A, Mo antisense oligonucleotides labeled with lissamine were applied immediately after ONS. A maximum-intensity projection of a deconvoluted Z-stack from a retina, 3 d after ONS, illustrates that RGCs and axons are labeled by retrograde Mo transport. B, C, Reggie-2 Ab immunostaining is present in control Mo-treated retinas which did not alter reggie expression (B). Reggie Mos reduced reggie-2 Ab staining of RGCs 7 d after ONS (C, images were taken with the same microscope settings). Scale bars, 50 μm. D, Regeneration was assessed by quantifying axons from mini-explants isolated 4 d after ONS. Retina pairs (21) (control- and reggie–Mo-treated eyes from the same fish) were analyzed. The mean number of axons per explant was normalized to the control retina, and relative outgrowth efficiency is shown.

Article Snippet: Anti-Reggie-1 [ESA (epidermal surface antigen)], anti-cdc42, and anti-Rac1 monoclonal antibodies (mAbs) were from BD Transduction Laboratories, anti-RhoA mAb, anti-Arp3 polyclonal antibody (pAB), and anti N-WASP mAb from Santa Cruz, anti-Ras mAb from Oncogene/Calbiochem.

Techniques: Labeling, Immunostaining, Expressing, Staining, Microscopy, Isolation

Journal: The Journal of Neuroscience

Article Title: Reggies/Flotillins Regulate Retinal Axon Regeneration in the Zebrafish Optic Nerve and Differentiation of Hippocampal and N2a Neurons

doi: 10.1523/JNEUROSCI.0870-09.2009

Figure Lengend Snippet: Reggie-1 knockdown impairs process formation in N2a cells. Cells transfected with either GL2 control siRNAs or a mix of reggie-1-specific siRNAs were stimulated with IGF-1, fixed 24 h after stimulation, and stained with phalloidin to visualize F-actin and cell morphology. Untreated and GL2 siRNA-transfected cells produced numerous filopodia (A, B), whereas reggie-1 downregulation (R1 siRNA) led to the formation of large lamellipodia (F). Significantly fewer cells formed neurites after reggie-1 siRNA transfection (E), which efficiently downregulated reggie-1 and reggie-2 expression compared with GL2 siRNA cells in Western blot experiments (G). D, N2a cells were simultaneously transfected with reggie-1 siRNA and the reggie-1 construct (R1–EGFP rescue) without siRNA-binding sites. A large proportion of the cells no longer exhibited the lamellipodia-rich phenotype but had instead many filopodia and neurites (D–F) much as control cells (A, B). Mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, Student's t test. Scale bars, 20 μm.

Article Snippet: Anti-Reggie-1 [ESA (epidermal surface antigen)], anti-cdc42, and anti-Rac1 monoclonal antibodies (mAbs) were from BD Transduction Laboratories, anti-RhoA mAb, anti-Arp3 polyclonal antibody (pAB), and anti N-WASP mAb from Santa Cruz, anti-Ras mAb from Oncogene/Calbiochem.

Techniques: Transfection, Staining, Produced, Expressing, Western Blot, Construct, Binding Assay

Journal: The Journal of Neuroscience

Article Title: Reggies/Flotillins Regulate Retinal Axon Regeneration in the Zebrafish Optic Nerve and Differentiation of Hippocampal and N2a Neurons

doi: 10.1523/JNEUROSCI.0870-09.2009

Figure Lengend Snippet: Reggie-1 is required for hippocampal neuron differentiation. Representative wide-field images of transfected hippocampal neurons. A, Cells transfected with control (GL2) siRNA differentiated normally (insert) having long thin axons (to the right) and dendritic arborizations. B, C, Note the short and thick protrusions, large lamellipodia, and broad dendrites of reggie-1 (R1) siRNA-treated cells. D, Cells simultaneously transfected with the reggie-1 rescue construct (R1–EGFP rescue) and reggie-1 siRNAs differentiated normally. Scale bars, 20 μm. Histogram in E shows the quantification of the cells transfected with control GL2 siRNA, with reggie-1 siRNAs, and simultaneously transfected with R1–EGFP rescue construct and reggie-1 siRNAs.

Article Snippet: Anti-Reggie-1 [ESA (epidermal surface antigen)], anti-cdc42, and anti-Rac1 monoclonal antibodies (mAbs) were from BD Transduction Laboratories, anti-RhoA mAb, anti-Arp3 polyclonal antibody (pAB), and anti N-WASP mAb from Santa Cruz, anti-Ras mAb from Oncogene/Calbiochem.

Techniques: Transfection, Construct

Journal: The Journal of Neuroscience

Article Title: Reggies/Flotillins Regulate Retinal Axon Regeneration in the Zebrafish Optic Nerve and Differentiation of Hippocampal and N2a Neurons

doi: 10.1523/JNEUROSCI.0870-09.2009

Figure Lengend Snippet: Reggie-1 downregulation affects Rho GTPase activation in N2a cells. N2a cells starved overnight, stimulated with 50 ng/ml IGF-1 for 5 min, and assayed for GTP-loading of small GTPases. The histograms, A–D, show changes in the intensity (relative to controls) in the respective Western blots (n = 4), which are exemplified below each histogram. Mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, Student's t test. Total Rac, cdc 42, RhoA, and Ras of crude lysates were used as loading control. A, B, In reggie-1 siRNA-treated cells, the activation patterns of Rac1 and cdc42 were significantly altered. IGF-1 application increased the activation of Rac1 in control-transfected cells, whereas siRNA-treated cells showed an elevated level of activated Rac1 without IGF-1, which increased further after IGF application. In contrast, cdc42 activity decreased during IGF application in control-transfected cells and remained at low levels in siRNA-transfected cells with or without IGF-1. C, RhoA activation increased during IGF application in control cells and further increased in reggie-1 siRNA-treated cells. D, Ras stimulation by IGF in control transfectants was absent in reggie–siRNA-treated cells.

Article Snippet: Anti-Reggie-1 [ESA (epidermal surface antigen)], anti-cdc42, and anti-Rac1 monoclonal antibodies (mAbs) were from BD Transduction Laboratories, anti-RhoA mAb, anti-Arp3 polyclonal antibody (pAB), and anti N-WASP mAb from Santa Cruz, anti-Ras mAb from Oncogene/Calbiochem.

Techniques: Activation Assay, Western Blot, Transfection, Activity Assay

Journal: The Journal of Neuroscience

Article Title: Reggies/Flotillins Regulate Retinal Axon Regeneration in the Zebrafish Optic Nerve and Differentiation of Hippocampal and N2a Neurons

doi: 10.1523/JNEUROSCI.0870-09.2009

Figure Lengend Snippet: Coimmunoprecipitation assays for the analysis of the complex of Arp3, N-WASP, and cortactin and phosphorylation of cofilin and cortactin in reggie siRNA-treated N2a cells. A, In IGF-1 stimulated control cells, Abs against N-WASP and cortactin coprecipitate Arp3. In reggie-1 siRNA-treated cells, the Arp3, N-WASP, cortactin complex is present at lower levels and is almost disrupted during IGF stimulation. B, The phosphorylation of cortactin at Tyr466 is unchanged after IGF-1 treatment of control cells but is strongly reduced during IGF-1 treatment of reggie-1 siRNA cells. C, The phosphorylation of cofilin at Ser3 slightly decreases during IGF stimulation but is increased in reggie-1 siRNA-treated cells with or without IGF-1.

Article Snippet: Anti-Reggie-1 [ESA (epidermal surface antigen)], anti-cdc42, and anti-Rac1 monoclonal antibodies (mAbs) were from BD Transduction Laboratories, anti-RhoA mAb, anti-Arp3 polyclonal antibody (pAB), and anti N-WASP mAb from Santa Cruz, anti-Ras mAb from Oncogene/Calbiochem.

Techniques:

Journal: The Journal of Neuroscience

Article Title: Reggies/Flotillins Regulate Retinal Axon Regeneration in the Zebrafish Optic Nerve and Differentiation of Hippocampal and N2a Neurons

doi: 10.1523/JNEUROSCI.0870-09.2009

Figure Lengend Snippet: Reggie-1 downregulation affects signaling in N2a cells. A, Activation of p38 was reduced in reggie siRNA-treated cells independently of IGF-1 stimulation in comparison with control transfectants. B, ERK1/2 activation was decreased in response to reggie siRNA. C, pFAK showed a significant activation during IGF-1 stimulation, and this stimulation was reduced in reggie-1 siRNA-treated cells. Total FAK was decreased in reggie-1 siRNA-treated cells. Total p38 (A), ERK1/2 (B), and GAPDH (C) were used as loading controls. Mean ± SEM; n = 4; *p < 0.05, **p < 0.01, ***p < 0.001, Student's t test. D, JNK, PKC, and PKB phosphorylation was not affected by reggie-1 siRNA-treatment.

Article Snippet: Anti-Reggie-1 [ESA (epidermal surface antigen)], anti-cdc42, and anti-Rac1 monoclonal antibodies (mAbs) were from BD Transduction Laboratories, anti-RhoA mAb, anti-Arp3 polyclonal antibody (pAB), and anti N-WASP mAb from Santa Cruz, anti-Ras mAb from Oncogene/Calbiochem.

Techniques: Activation Assay

Journal: Journal of Cell Science

Article Title: Pro-cathepsin D interacts with the extracellular domain of the ? chain of LRP1 and promotes LRP1-dependent fibroblast outgrowth

doi: 10.1242/jcs.070938

Figure Lengend Snippet: COS cells transfected with cath-D (panel a), or cath-D + LRP1β (panel b) were unwashed and lysed 48 h post-transfection. Lysates were submitted to sucrose density ultracentrifugation. Fractions were analyzed for anti-LRP1β (top panels) or anti-cath-D (middle panels) by immunoblotting. Cell lysates (100 μg) from cath-D-transfected (CE1) and, cath-D + LRP1β-co-transfected COS cells (CE2), were analyzed by immunoblotting. CE2 was loaded onto both gels (panels a and b) as an internal control for cath-D or LRP1β enrichment relative to film exposure. Brackets indicate pro-cath-D and its neo-processed forms. Flotillin was used as a marker of raft fractions, and TfR as a marker of non-raft fractions (bottom panels). Each fraction was dot-blotted to detect ganglioside GM1 (bottom panels). K, molecular mass in kilodaltons.

Article Snippet: Control IgG1 MOPC-21 monoclonal antibody was purchased from Abcam, anti-β actin polyclonal antibody from Sigma, anti-human Flotillin-1 monoclonal antibody from BD Biosciences, and anti-human Transferrin receptor monoclonal antibody from Zymed.

Techniques: Transfection, Western Blot, Marker

Journal: Molecular & Cellular Proteomics : MCP

Article Title: HIV-1 Nef Changes the Proteome of T Cells Extracellular Vesicles Depleting IFITMs and Other Antiviral Factors

doi: 10.1016/j.mcpro.2023.100676

Figure Lengend Snippet:

Article Snippet: Anti-Flotillin1 (ab41927) , (Abcan).

Techniques: