Journal: Frontiers in cell and developmental biology

Article Title: Differential Effects of APOE Genotype on MicroRNA Cargo of Cerebrospinal Fluid Extracellular Vesicles in Females With Alzheimer's Disease Compared to Males.

doi: 10.3389/fcell.2022.864022

Figure Lengend Snippet: FIGURE 3 | SEC qEV single 35 nm columns are optimal for separating CSF EVs. Pools of SEC fractions (Fxs): 1–5 (column void volume), 6–9, 10–13, and 14–17 were generated using either the qEV Single 35 nm or 70 nm columns. (A) Equal concentration loading of protein lysate (0.1 µg) from SEC pools immunoblotted for flotillin and CD81. (B) Equal volume loading of protein lysate (37 µL) from SEC pools immunoblotted for APOA1 and albumin. (C) Size and concentration histograms of Fxs 6–9 generated using either the qEV Single 35 nm (white) or 70 nm (black) columns acquired by tunable resistive pulse sensing (TRPS).

Article Snippet: The following antibodies were used for immunoblotting: albumin 1:1,000 (#4929, Cell Signaling Technology, Danvers, MA), APOA1 (12C8) 1:200 (sc-080551, Santa Cruz Biotechnology, Dallas, TX), APOE 1:2000 (50A-G1A, Academy Bio-medical Company, Inc., Houston, TX), AnnV 1: 5,000 (GTX103250, GeneTex, Irvine, CA), CD9 (C-4) 1:200 (sc13118, Santa Cruz Biotechnology), CD11b 1:1,000 (ab133357, Abcam, Cambridge, United Kingdom), CD63 1:1,000 (ab134045, Abcam), CD81 (B-11) 1:100 (sc-166029, Santa Cruz Biotechnology), flotillin 1:10,000 (ab133497, Abcam), GLAST 1:500 (NB100-1869, Novus Biologicals, Littleton, CO), NCAM1 1:125 (NBP2-38452, Novus Biologicals), SYP 1:1,000 (#36406, Cell Signaling Technology), TMEM119 (#66948-1-Ig, Proteintech, Rosemont, IL), and TSG101 1:1,000 (ab125011, Abcam).

Techniques: Generated, Concentration Assay, Tunable Resistive Pulse Sensing

Journal: Frontiers in cell and developmental biology

Article Title: Differential Effects of APOE Genotype on MicroRNA Cargo of Cerebrospinal Fluid Extracellular Vesicles in Females With Alzheimer's Disease Compared to Males.

doi: 10.3389/fcell.2022.864022

Figure Lengend Snippet: FIGURE 5 | CSF EVs isolated by SEC are enriched for exosome and MV markers. (A) Equal volume loading of protein lysate (37 µL) from pools of SEC Fractions (Fxs): 1–5 (column void volume), 6–9, 10–13, and 14–17 stained for total protein, and immunoblotted for albumin, APOA1, and APOE. (B) Equal concentration loading of protein lysate (0.1 µg) from pools of Fxs 1–5, 6–9, 10–13, and 14–17 immunoblotted for CD9, CD63, CD81, flotillin, TSG101, AnnV, SYP, NCAM-1, GLAST, CD11b, and TMEM119. (C) Equal concentration loading of (1 µg) from pools of Fxs 1–5, 6–9, 10–13, and 14–17 immunoblotted for SYP and CD11b. Postmortem human cerebral cortex protein lysate (0.1 µg and 1 µg) was run as a positive control for each gel ((A-C): Brain).

Article Snippet: The following antibodies were used for immunoblotting: albumin 1:1,000 (#4929, Cell Signaling Technology, Danvers, MA), APOA1 (12C8) 1:200 (sc-080551, Santa Cruz Biotechnology, Dallas, TX), APOE 1:2000 (50A-G1A, Academy Bio-medical Company, Inc., Houston, TX), AnnV 1: 5,000 (GTX103250, GeneTex, Irvine, CA), CD9 (C-4) 1:200 (sc13118, Santa Cruz Biotechnology), CD11b 1:1,000 (ab133357, Abcam, Cambridge, United Kingdom), CD63 1:1,000 (ab134045, Abcam), CD81 (B-11) 1:100 (sc-166029, Santa Cruz Biotechnology), flotillin 1:10,000 (ab133497, Abcam), GLAST 1:500 (NB100-1869, Novus Biologicals, Littleton, CO), NCAM1 1:125 (NBP2-38452, Novus Biologicals), SYP 1:1,000 (#36406, Cell Signaling Technology), TMEM119 (#66948-1-Ig, Proteintech, Rosemont, IL), and TSG101 1:1,000 (ab125011, Abcam).

Techniques: Isolation, Staining, Concentration Assay, Positive Control

Journal: Cell reports

Article Title: MAL2 mediates the formation of stable HER2 signaling complexes within lipid raft-rich membrane protrusions in breast cancer cells

doi: 10.1016/j.celrep.2021.110160

Figure Lengend Snippet: (A) Immunofluorescence staining for HER2 and cholera toxin B in MCF10A and SKBR3 cells. Scale bars represent 10 μm. (B) Lipid raft areas on the cell surface, with quantification in the bar graph on the right. (C) Flotillin1 (FLOT1), MAL, and MAL2 mRNA expression in different breast cancer cell lines as assessed by quantitative PCR (n = 3). (D) RNA-seq analysis of FLOT1 and MAL2 expression in normal breast tissue (n = 112) and HER2-positive breast tumors (n = 160) represented in The Cancer Genome Atlas database. (E) Uniform Manifold Approximation and Projection (UMAP) plots of breast cancer single-cell RNA-seq data (GEO: GSE75688, left) and co-expression pattern of HER2, FLOT1, MAL, and MAL2 in cells from cluster 2. (F) Distribution of MAL, FLOT1, MAL2, and HER2 expression level for each cell in cluster 2. (G) MAL2 ATAC-seq peak clusters in SKBR3, MCF10A, MCF7, and MDA-MB-231 cell lines. In the bar graphs, the bars represent the mean ± SEM. **p <0.01, ***p <0.001, ****p <0.0001. These results are representative of three independent experiments.

Article Snippet: Constructs encoding flag-flotillin1 (RC200231) and flag-MAL2 (RC203862) are commercially available from OriGene (Rockville, MD).

Techniques: Immunofluorescence, Staining, Expressing, Real-time Polymerase Chain Reaction, RNA Sequencing

Journal: Cell reports

Article Title: MAL2 mediates the formation of stable HER2 signaling complexes within lipid raft-rich membrane protrusions in breast cancer cells

doi: 10.1016/j.celrep.2021.110160

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Constructs encoding flag-flotillin1 (RC200231) and flag-MAL2 (RC203862) are commercially available from OriGene (Rockville, MD).

Techniques: Recombinant, Real-time Polymerase Chain Reaction, RNA Sequencing, Sequencing, Microarray, Control, Software, Imaging, Light Microscopy

Journal: Brain Communications

Article Title: Antibodies against the flotillin-1/2 complex in patients with multiple sclerosis

doi: 10.1093/braincomms/fcad109

Figure Lengend Snippet: Immunocytochemistry of anti-FLOT1/2 antibodies (commercial slides) . Commercial biochips (Euroimmun) containing FLOT-1/2 transfected HEK293 cells ( A , C ) or non-transfected HEK293 cells ( B , D ); incubated with serum from MS Patient 1 ( A , B ) and from a healthy donor ( C , D ). Patient 1 showed strong IgG reactivity against co-transfected cells ( A ) in comparison with non-transfected cells ( B ) and with the negative control ( C ).

Article Snippet: Briefly, mammalian expression vectors encoding human FLOT1 (SC319463, Origene) and FLOT2 (EX-H1820-M02, Genecopoeia) were transfected into HEK293 cells using Lipofectamine 2000.

Techniques: Immunocytochemistry, Transfection, Incubation, Comparison, Negative Control

Journal: Brain Communications

Article Title: Antibodies against the flotillin-1/2 complex in patients with multiple sclerosis

doi: 10.1093/braincomms/fcad109

Figure Lengend Snippet: Immunocytochemistry of anti-FLOT1/2 antibodies (in house transfection) . HEK293 cells co-transfected with mammalian-expression vectors encoding human FLOT1 and FLOT2 using Lipofectamine 2000; double-stained with serum ( B , E ) and with commercial antibody against FLOT1 ( A , D ). MS patient’s IgG bind to co-transfected cells ( B ) and colocalize with FLOT1 ab ( C ); in contrast with the healthy control ( E ) that doesn’t show any reactivity against FLOT-1/2 antibodies ( F ).

Article Snippet: Briefly, mammalian expression vectors encoding human FLOT1 (SC319463, Origene) and FLOT2 (EX-H1820-M02, Genecopoeia) were transfected into HEK293 cells using Lipofectamine 2000.

Techniques: Immunocytochemistry, Transfection, Expressing, Staining, Control

Journal: Brain Communications

Article Title: Antibodies against the flotillin-1/2 complex in patients with multiple sclerosis

doi: 10.1093/braincomms/fcad109

Figure Lengend Snippet: Immunoadsorption of anti-FLOT1/2 antibodies . HEK293 cells co-transfected with mammalian-expression vectors encoding human FLOT1 and FLOT2 double-stained with serum immunoadsorbed with non-transfected cells ( A ), FLOT1-transfected cells ( D ), FLOT2-transfected cells ( G ) or FLOT1 and FLOT2 co-transfected cells ( J ); and with commercial antibody against FLOT1 ( B , E , K ) or FLOT2 ( H ). Reactivity against the FLOT-1/2 complex was lost after serum pre-adsorption with HEK cells co-expressing FLOT1 and FLOT2 ( L ), but not after pre-adsorption with cells transfected with FLOT1 ( F ) or FLOT2 ( I ) alone or with non-transfected HEK cells ( C ).

Article Snippet: Briefly, mammalian expression vectors encoding human FLOT1 (SC319463, Origene) and FLOT2 (EX-H1820-M02, Genecopoeia) were transfected into HEK293 cells using Lipofectamine 2000.

Techniques: Transfection, Expressing, Staining, Adsorption