flotillin  (Novus Biologicals)

Journal: Journal of Extracellular Biology

Article Title: Dysregulation of Extracellular Vesicle Concentration, MicroRNAs, and Surface Proteins in Patients With Niemann‐Pick Disease Type C

doi: 10.1002/jex2.70136

Figure Lengend Snippet: SEC enriches for EVs from fibroblast conditioned media . ( A ) EV enrichment method for fibroblast conditioned media. ( B, C ) Immunoblots of individual ( B ) or pooled ( C ) SEC fractions from CTL fibroblast media show enrichment for EV proteins Flotillin‐1, CD63, and CD81, without detectable levels of other proteins (total protein stain) or cell lysate protein GM130. ( D ) MRPS histogram shows the distribution of particles in pooled EV fractions isolated from CTL fibroblast media. Mean particle count, size, and coefficient of variation are listed.

Article Snippet: Primary antibodies included NPC1 (EPR5209) 1:1000 (134113, Abcam); CD63 (H5C6) 1:5,000 (556019, BD Biosciences); CD81 (B‐11) 1:100 (sc‐166029, Santa Cruz Biotechnology); Flotillin‐1 1:10,000 (ab133497, Abcam); GM130 1:1000 (53420SS, Novus Biologicals); and Lamp1 (D2D11) 1:1000 (9091S XP, Cell Signaling Technology).

Techniques: Western Blot, Staining, Isolation

Journal: Journal of Extracellular Biology

Article Title: Dysregulation of Extracellular Vesicle Concentration, MicroRNAs, and Surface Proteins in Patients With Niemann‐Pick Disease Type C

doi: 10.1002/jex2.70136

Figure Lengend Snippet: SEC enriches for EVs from human CSF . ( A ) EV enrichment method for CSF. ( B, C ) Immunoblots of individual ( B ) or pooled ( C ) SEC fractions from CTL CSF show enrichment for EV proteins Flotillin‐1 and CD81 without detectable levels of bulk CSF proteins (total protein stain, albumin, ApoA1). ( D ) MRPS histogram shows the distribution of particles in pooled EV fractions from CTL CSF. Mean particle count, size, and coefficient of variation are listed. ( E ) TEM of representative NPC CSF EV sample. Black arrows indicate particles of ∼100 nm; white arrows indicate particles of ∼50 nm.

Article Snippet: Primary antibodies included NPC1 (EPR5209) 1:1000 (134113, Abcam); CD63 (H5C6) 1:5,000 (556019, BD Biosciences); CD81 (B‐11) 1:100 (sc‐166029, Santa Cruz Biotechnology); Flotillin‐1 1:10,000 (ab133497, Abcam); GM130 1:1000 (53420SS, Novus Biologicals); and Lamp1 (D2D11) 1:1000 (9091S XP, Cell Signaling Technology).

Techniques: Western Blot, Staining

Journal: Journal of Virology

Article Title: Glycosylated NS3/NS3A protein of bluetongue virus facilitates efficient viral egress via lipid raft anchoring

doi: 10.1128/jvi.02144-25

Figure Lengend Snippet: N-linked glycosylation of NS3/NS3A facilitates its raft-enriched membrane association and efficient BTV release. ( A ) Representative confocal images of HeLa cells transfected with NS3/NS3A WT or the N150Q mutant. Cells were fixed and stained 20 h post-transfection. NS3/NS3A was detected using anti-NS3/NS3A (red), and lipid raft-enriched membrane domains were labeled with anti-flotillin-1 (green). Scale bars, 5 μm. ( B and C ) Confocal images of HeLa cells expressing NS3/NS3A treated with trifluoperazine (TFP) ( B ) or methyl-β-cyclodextrin (MβCD) ( C ). Cells were fixed and stained 20 h post-transfection. NS3/NS3A is shown in red and flotillin-1 in green. Fluorescence intensity profiles were analyzed along the indicated line scans. Scale bars, 5 μm. ( D ) Localization of NS3/NS3A at the plasma membrane of HeLa cells with or without MβCD or TFP treatment. The plasma membrane was stained using WGA-Alexa Fluor 488 (green), and NS3/NS3A was visualized in red. Scale bars, 5 μm. ( E ) Quantification of plasma membrane (PM) localization from panel D, shown as the ratio of PM-associated NS3/NS3A fluorescence intensity (defined by WGA staining) to total cellular NS3/NS3A intensity. Values were normalized to vehicle-treated controls and are presented as mean ± SD. Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons test (** P < 0.01, **** P < 0.0001). ( F ) MDOK or HEK-293T cells were infected with BTV-20 WT or BTV-20 N150Q at an MOI of 5. At 6 h post-infection, the cells were treated with MβCD or TFP, and viral titers were determined at 12 h post-infection. Data represent mean ± SD ( n = 3 biological replicates). Statistical significance was determined by two-way ANOVA with Šídák’s multiple comparisons test (ns, not significant; *** P < 0.001; **** P < 0.0001). ( G ) Analysis of BTV-20 release efficiency following raft disruption. MDOK cells were infected with BTV-20 (MOI = 5) and then treated with MβCD or TFP. Extracellular and intracellular viral titers were determined at the indicated time points. Release efficiency was calculated as [extracellular titer/(extracellular + intracellular titer)] × 100%. Data are presented as mean ± SD ( n = 3 biological replicates). Statistical significance was determined using two-way ANOVA with Šídák’s multiple comparisons test (ns, not significant; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001). ( H ) A total of 1,136 host proteins were identified by quantitative proteomics, of which 102 proteins were enriched in the NS3/NS3A WT interactome relative to the N150Q mutant (log₂ FC > 4 or unique to WT). Highlighted are the top eight candidates. ( I ) Gene Ontology (GO) enrichment analysis of proteins enriched in the NS3/NS3A WT interactome. Significantly overrepresented terms are shown for the biological process, cellular component, and molecular function categories. Bars represent −log₁₀ ( P value). ( J ) Sucrose gradient fractionation followed by immunoblotting of lysates from transfected HEK-293T cells, or from infected MDOK cells. DRM (raft-enriched) fractions correspond to fractions 5–12, whereas detergent-soluble membrane (DSM) fractions correspond to fractions 13–20. ( K ) Co-immunoprecipitation analysis of NS3/NS3A WT , NS3/NS3A N150Q , and FLNA. Cells were transfected with NS3/NS3A variants, followed by Co-IP.

Article Snippet: Commercial antibodies used in this study included anti-FLAG (DYKDDDDK) monoclonal antibody (1:1,000 for immunofluorescence assay [IFA], 1:10,000 for western blotting [WB]; 66008-4-Ig, Proteintech), anti-HA polyclonal antibody (1:100 for IFA, 1:1,000 for WB; 51064-2-AP, Proteintech), anti-β-actin monoclonal antibody (1:10,000 for WB; 66009-1-Ig, Proteintech), anti-Calnexin polyclonal antibody (1:200 for IFA; 10427-2-AP, Proteintech), anti-Syntaxin 6 polyclonal antibody (1:200 for IFA; 10841-1-AP, Proteintech), anti-Flotillin 1 monoclonal antibody (1:100 for IFA; 67968-1-Ig, Proteintech), and anti-Filamin A (FLNA) monoclonal antibody (1:1,000 for WB; 67133-1-Ig, Proteintech).

Techniques: Glycoproteomics, Membrane, Transfection, Mutagenesis, Staining, Labeling, Expressing, Fluorescence, Clinical Proteomics, Infection, Disruption, Quantitative Proteomics, Fractionation, Western Blot, Immunoprecipitation, Co-Immunoprecipitation Assay