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

Journal: Life Science Alliance

Article Title: Systematic identification of ALK substrates by integrated phosphoproteome and interactome analysis

doi: 10.26508/lsa.202101202

Figure Lengend Snippet: Identified phosphorylation sites of ALK substrate candidates.

Article Snippet: ANXA2, APLP2, CNP, CYFIP2, EMD, ESYT1, ESYT2, FAF2, FLOT1, GMDS, LASP1, PIN4, PTPN11, PXN, PTPRS, PXN, SHC1, SMAD1, and UBXN6 were purchased from Origene.

Techniques:

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: Molecules and Cells

Article Title: Altered Proteome of Extracellular Vesicles Derived from Bladder Cancer Patients Urine

doi: 10.14348/molcells.2018.2110

Figure Lengend Snippet: The circular particles with around 120nm diameter were visualized by TEM and NTA, which were further analyzed with immunological assays to show positive expression of specific vesicular molecules such as Alix, CD63, tumor susceptibility gene-101 (TSG-101) and flotillin-1.

Article Snippet: Antibodies which were used for western blot and flow cytometry are as follow; anti-human CD63 (clone H5C6, mouse monoclonal, BD Biosciences, USA), anti-human TSG101 (clone EPR7130(B), rabbit monoclonal, Millipore, USA), anti-human flotillin-1 (Clone 18/Flotillin-1, mouse polyclonal, BD Biosciences, USA), anti-human HSP70 (clone W27, mouse monoclonal, BioLegend, USA) and anti-human Alix (clone 3A9, mouse polyclonal, Cell Signaling, USA).

Techniques: Expressing

Journal: Journal of Alzheimer's disease : JAD

Article Title: Neuronal Exosome-Derived Human Tau is Toxic to Recipient Mouse Neurons in vivo

doi: 10.3233/JAD-180776

Figure Lengend Snippet: Exosomes derived from neuronally-differentiated, human induced pluripotent stem cells (NiPSCEs) contain human tau species. Representative NTA plot of averaged size/concentration for exosomes derived from tau-RD-LM-YFP conditioned media from induced pluripotent stem cells (NiPSCEs) (A). Non-exosome (non-exo) and exosomal (exo) fractions were probed against the human recombinant tau protein using MC-1 and PHF1 antibodies. Representative western blots demonstrate that MC-1 and PHF1 immunoreactive tau is only detected in the exosomal fraction derived from NiPSCEs. Exosome surface marker, Flotillin-1, was used as a loading control (B).

Article Snippet: 312–428, cross reacts with human flotillin-1; 1:100, 610821, BD Transduction Laboratories, San Jose, CA).

Techniques: Derivative Assay, Concentration Assay, Recombinant, Western Blot, Marker