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  • 92
    Millipore anti flotillin 1 antibody
    Anti Flotillin 1 Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 41 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson flotillin 2
    Flotillin upregulation stimulates AXL endocytosis towards non-degradative late endosomes and promotes its stabilization. A) AXL knock-down in MCF10AF1F2 cells decreases ZEB1 and N-cadherin expression . MCF10AmCh and MCF10AF1F2 cells were transfected with control siRNA (CTL) or against AXL and probed by western blotting with antibodies for the indicated proteins. The quantification results are the mean ± SEM of 4 independent experiment, and are expressed as fold-increase compared with control (MCF10AmCh). B) ZEB1 nuclear localization analyzed by immunocytochemistry and co-staining with Hoechst (Hst) and C) E-cadherin distribution analyzed by immunocytochemistry in MCF10AmCh and MCF10AF1F2 cells transfected with control (CTL) or anti-AXL siRNAs. Results shown are representative of 4 independent experiments. Scale bar: 10 µm. C) Flotillin-oligomerization induced by a CRY2-CIBN-based optogenetic approach promotes co-clustering of AXL . Still images from a TIRF-microscopy movie of a MCF10A cell that co-expresses CRY2-mCitrin, flotillin 2-CIBN-mCherry, and AXL-Halo labeled with Halo-Tag-Janelia Fluor 646. Representative image of a whole cell 15s after starting the 488 nm illumination to perform CRY2 binding to flotillin 2-CIBN-mCh and subsequent oligomerization (see also supplementary Fig. S6 for the details regarding the validation of the constructs and the optogenetic approach). Magnified images from the boxed area taken before and after 488 nm illumination. Arrowheads indicate clusters of CRY2-mCitrin and flotillin 2-CIBN-mCherry where AXL-Halo accumulates (see also video 4). Scale bars: 10 µm for the main image and 2 µm for the images from the boxed area. D) Co-accumulation of AXL and <t>flotillin</t> 2 at the PM prior to their co-endocytosis . Still images of a time-lapse series of one MCF10AF1F2 cell that expresses AXL-GFP to show flotillin 2/AXL co-localization at the PM and their co-endocytosis at PM sites (showed by arrowheads). Kymographs show the temporal variation of the GFP and mCherry signals along two line-scans (blue at the PM endocytic site, and yellow close to the PM endocytic site). Graphs show the intensity variation of both signals along the dotted white lines on the kymographs. The left kymograph shows the accumulation of AXL-GFP and flotillin 2-mCherry at the PM followed by a sudden concomitant drop of both signals (around 83s, indicated by a star). The right kymograph shows that in the meantime, both AXL-GFP and flotillin 2-mCherry signals remained stable at the PM outside and close to this endocytic site (See also video 5). E) Flotillin 1 co-precipitates with AXL. AXL was immunoprecipitated from MCF10AF1F2 cell lysates, and flotillin 1 co-immunoprecipitation was assessed by immunoblotting. Control immunoprecipitation was performed with non-relevant immunoglobulins (Ig). F) Flotillin upregulation accelerates AXL endocytosis. Kinetics of AXL internalization in MCF10AmCh and MCF10AF1F2 cells (left), and in MDA-MB-231shLuci and MDA-MB-231shFlot2 cells (right). Surface proteins were labeled with biotin at 4°C, and cells were incubated at 37°C for the indicated times to allow endocytosis. AXL presence in the internalized biotinylated fraction was probed by western blotting using relevant antibodies and quantified as the percentage of the maximum level of internalized protein (reached at 12 and 8 min, respectively, in MCF10AmCh and MCF10AF1F2 cells). Results are expressed as the mean ± SEM of 4 independent experiments. The AXL internalization rates (dashed lines) were 8.48 ± 1.28 and 12.43 ± 0.48 %/min in MCF10AmCh and MCF10AF1F2 cells, respectively, revealing a significant 1.45-fold increase (p=0.0088) upon flotillin upregulation. Comparison of the AXL internalization rates (dashed lines) in MDA-MB-231shLuci and MDA-MB-231shFlot2 cells showed a significant 3.66-fold decrease (p=0.0009) upon flotillin downregulation. Results are expressed as the percentage of the maximum level of surface labeled AXL at T0 and are the mean ± SEM of 4 independent experiments. G) Flotillin upregulation promotes AXL protein level increase . Cell lysates from MCF10AmCh and MCF10AF1F2 cells were probed by western blotting with antibodies against AXL phosphorylated on Y702, AXL and tubulin. Results are expressed as fold-increase compared with MCF10AmCh cells and are the mean ± SEM of 6 independent experiments. H) Flotillin upregulation does not increase AXL mRNA level. RT-qPCR analysis of AXL expression in MCF10AmCh and MCF10AF1F2 cells. Results are expressed relative to the level in MCF10AmCh cells and are the mean ± SEM of 4 independent experiments. I) Flotillin upregulation stabilizes AXL. MCF10A and MCF10AF1F2 cells were incubated with cycloheximide (CHX, 100µg/ml) and cell lysates collected at the indicated time points. AXL and tubulin levels were assessed by western blotting. The graph shows the normalized AXL levels in each cell line during CHX incubation. CHX treatment was stopped after 9 hours because MCF10Amch cells did not survive longer treatment. *P
    Flotillin 2, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 355 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson antibodies flotillin 1
    Knockdown of <t>flotillin-1</t> or flotillin-2 results in altered focal adhesion morphology during cell spreading. HeLa cells were transfected with the indicated siRNAs. The cells were detached, kept in suspension, and then seeded on fibronectin for 25 min. Focal adhesions were visualized by vinculin staining and filamentous actin by phalloidin staining. Scale bar: 10 µm, the same magnification for all images.
    Antibodies Flotillin 1, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc flotillin 1
    Verification of EV isolation. Laemmli and TRIzol©-extracted samples were interrogated by Western blot for the expression of the canonical EV markers HSP-70, CD63, <t>Flotillin-1,</t> CD81 and CD9. MCF-7 (7), MCF-10A (10A) and MDA-MB-231 (231) cell EVs were examined. HSP-70 (top), CD63 (middle) and Flotillin-1 (bottom) were detected in the isolated EVs.
    Flotillin 1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 731 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology flotillin 1
    <t>Flotillin</t> depletion increases diffusion mobility rates of CFP-HA-DAT
    Flotillin 1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 234 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam flotillin 1
    GPCR activation triggers MVB–PM fusion in HeLa cells via SNAP23-Ser110 phosphorylation. (a) Fusion activity of histamine-stimulated cells nontreated or treated with the Gα q inhibitor UBO-QIC (1 µM). n ≥ 24 cells per condition. (b) Basal fusion activity in cells treated with PKC inhibitors GÖ6976 (1 µM) or GÖ6983 (1 µM). n ≥ 11 cells per condition. (c) Fusion activity of histamine-stimulated cells nontreated or preincubated with GÖ6983 (1 µM). n ≥ 11 cells per condition. (d) Schematic representation of SNAP23 with SNARE motifs, a membrane-anchoring domain (M), and all phosphosites with the posphosite targeted by histamine stimulation (Ser110) in bold. (e) Fusion activity of histamine-stimulated cells transfected with WT SNAP23, phosphomutant SNAP23-S110A, or phosphomimic SNAP23-S110D. n ≥ 16 cells per condition. (f) Left: fusion activity of CD63-, CD81-, and CD9-pHluorin HeLa cells cotransfected with SNAP23 WT or SNAP23-S110A. n ≥ 16 cells per condition. Western blot on exosomes isolated from SNAP23-WT and SNAP23-S110A HeLa cells labeled for CD63, CD9, CD81, <t>flotillin-1,</t> and syntenin-1. (g) Schematic representation of the histamine-stimulated pathway leading to exosome release as identified by phosphoproteomics and specific inhibitors. Blue-rimmed proteins represent the putative pathway implicated by both experiments. The IP3–Ca 2+ pathway is represented in gray as a direct link with MVB–PM fusion is missing. **, P
    Flotillin 1, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 244 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson mouse anti flotillin 1
    Dsg3 and other desmosomal proteins are membrane raft associated. Primary human keratinocytes were grown to confluence and switched to high calcium media for 16–18 hrs. Following detergent extraction (1% Triton X-100) and ultra-centrifugation on a 5–40% sucrose gradient, 12 fractions were sequentially removed from the gradient and processed via western blot. Dsg3 partitions to the buoyant raft fractions (DRMs, detergent resistant membranes) as indicated by the positive controls <t>flotillin-1</t> and caveolin-1, and negative control calnexin. Desmosomal components plakoglobin (PG) and plakophilin 2 (pkp-2) were also found to be raft associated. E-cadherin, a classical cadherin of adherens junctions, is not enriched in membrane rafts.
    Mouse Anti Flotillin 1, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 89/100, based on 173 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology anti flotillin 1
    Characterization of exosomes ( A ) Representative electron microscopy image of exosomes. ( B ) Western blot analysis of CD63, CD9 and β-actin in exosomes and CNE-2 cells. <t>Flotillin-1</t> was used as a loading control. ( C ) The number of exosomes was counted in 10 fields under TEM (transmission electron microscope).
    Anti Flotillin 1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 91/100, based on 79 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Syntaxin flotillin 2
    Non-raft association of PS1 and APP CTFs in embryonic mouse brain Raft and non-raft distribution of PS1, nicastrin, APP, <t>flotillin-2,</t> calnexin, and γ-adaptin in flotation density gradient fractions from E15.5 embryonic brains were analyzed by Western blotting. Note the predominant non-raft localization of PS1, nicastrin, and APP CTFs. mat , mature; imm , immature.
    Flotillin 2, supplied by Syntaxin, used in various techniques. Bioz Stars score: 91/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti flotillin 1
    Non-raft association of PS1 and APP CTFs in embryonic mouse brain Raft and non-raft distribution of PS1, nicastrin, APP, <t>flotillin-2,</t> calnexin, and γ-adaptin in flotation density gradient fractions from E15.5 embryonic brains were analyzed by Western blotting. Note the predominant non-raft localization of PS1, nicastrin, and APP CTFs. mat , mature; imm , immature.
    Anti Flotillin 1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti flotillin
    Non-raft association of PS1 and APP CTFs in embryonic mouse brain Raft and non-raft distribution of PS1, nicastrin, APP, <t>flotillin-2,</t> calnexin, and γ-adaptin in flotation density gradient fractions from E15.5 embryonic brains were analyzed by Western blotting. Note the predominant non-raft localization of PS1, nicastrin, and APP CTFs. mat , mature; imm , immature.
    Anti Flotillin, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti flotillin 1 antibody
    Non-raft association of PS1 and APP CTFs in embryonic mouse brain Raft and non-raft distribution of PS1, nicastrin, APP, <t>flotillin-2,</t> calnexin, and γ-adaptin in flotation density gradient fractions from E15.5 embryonic brains were analyzed by Western blotting. Note the predominant non-raft localization of PS1, nicastrin, and APP CTFs. mat , mature; imm , immature.
    Anti Flotillin 1 Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 91/100, based on 38 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson anti flotillin 1 monoclonal antibody
    Localization and cytotoxicity of VVH are not affected by SMase. CHO cells were incubated with (+) or without (−) 100 mU/ml SMase at 37°C for 1 h. After incubation, the cells were washed twice with DMEM and incubated with 5 µg/ml VVH at 37°C for 15 min. Cells were lysed, and fractionated. VVH, <t>flotillin-1</t> (flt-1), and transferrin receptor (TfR) were detected by western blotting using specific antibodies.
    Anti Flotillin 1 Monoclonal Antibody, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 85/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti flotillin 2
    Tat palmitoylation is performed by DHHC-20. a HEK 293 T cells were cotransfected (1/5) with the indicated myc-tagged human DHHC and Tat. Tat palmitoylation was then assessed using the acyl-RAC technique, UC unbound control, BC bound control, UH unbound hydroxylamine, BH bound hydroxylamine. Palmitoylated Tat is present in the BH fraction. Palmitoylation was calculated as BH/(BH + UH)-BC/(BC + UC), and <t>flotillin-2</t> was used as a positive control. The graph shows mean ± SEM of n = 3 independent experiments, *** p
    Anti Flotillin 2, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher flotillin 2
    <t>Flotillin</t> depletion increases diffusion mobility rates of CFP-HA-DAT
    Flotillin 2, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti flotillin 1 antibody epr6041
    <t>Flotillin</t> depletion increases diffusion mobility rates of CFP-HA-DAT
    Anti Flotillin 1 Antibody Epr6041, supplied by Abcam, used in various techniques. Bioz Stars score: 97/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Flotillin upregulation stimulates AXL endocytosis towards non-degradative late endosomes and promotes its stabilization. A) AXL knock-down in MCF10AF1F2 cells decreases ZEB1 and N-cadherin expression . MCF10AmCh and MCF10AF1F2 cells were transfected with control siRNA (CTL) or against AXL and probed by western blotting with antibodies for the indicated proteins. The quantification results are the mean ± SEM of 4 independent experiment, and are expressed as fold-increase compared with control (MCF10AmCh). B) ZEB1 nuclear localization analyzed by immunocytochemistry and co-staining with Hoechst (Hst) and C) E-cadherin distribution analyzed by immunocytochemistry in MCF10AmCh and MCF10AF1F2 cells transfected with control (CTL) or anti-AXL siRNAs. Results shown are representative of 4 independent experiments. Scale bar: 10 µm. C) Flotillin-oligomerization induced by a CRY2-CIBN-based optogenetic approach promotes co-clustering of AXL . Still images from a TIRF-microscopy movie of a MCF10A cell that co-expresses CRY2-mCitrin, flotillin 2-CIBN-mCherry, and AXL-Halo labeled with Halo-Tag-Janelia Fluor 646. Representative image of a whole cell 15s after starting the 488 nm illumination to perform CRY2 binding to flotillin 2-CIBN-mCh and subsequent oligomerization (see also supplementary Fig. S6 for the details regarding the validation of the constructs and the optogenetic approach). Magnified images from the boxed area taken before and after 488 nm illumination. Arrowheads indicate clusters of CRY2-mCitrin and flotillin 2-CIBN-mCherry where AXL-Halo accumulates (see also video 4). Scale bars: 10 µm for the main image and 2 µm for the images from the boxed area. D) Co-accumulation of AXL and flotillin 2 at the PM prior to their co-endocytosis . Still images of a time-lapse series of one MCF10AF1F2 cell that expresses AXL-GFP to show flotillin 2/AXL co-localization at the PM and their co-endocytosis at PM sites (showed by arrowheads). Kymographs show the temporal variation of the GFP and mCherry signals along two line-scans (blue at the PM endocytic site, and yellow close to the PM endocytic site). Graphs show the intensity variation of both signals along the dotted white lines on the kymographs. The left kymograph shows the accumulation of AXL-GFP and flotillin 2-mCherry at the PM followed by a sudden concomitant drop of both signals (around 83s, indicated by a star). The right kymograph shows that in the meantime, both AXL-GFP and flotillin 2-mCherry signals remained stable at the PM outside and close to this endocytic site (See also video 5). E) Flotillin 1 co-precipitates with AXL. AXL was immunoprecipitated from MCF10AF1F2 cell lysates, and flotillin 1 co-immunoprecipitation was assessed by immunoblotting. Control immunoprecipitation was performed with non-relevant immunoglobulins (Ig). F) Flotillin upregulation accelerates AXL endocytosis. Kinetics of AXL internalization in MCF10AmCh and MCF10AF1F2 cells (left), and in MDA-MB-231shLuci and MDA-MB-231shFlot2 cells (right). Surface proteins were labeled with biotin at 4°C, and cells were incubated at 37°C for the indicated times to allow endocytosis. AXL presence in the internalized biotinylated fraction was probed by western blotting using relevant antibodies and quantified as the percentage of the maximum level of internalized protein (reached at 12 and 8 min, respectively, in MCF10AmCh and MCF10AF1F2 cells). Results are expressed as the mean ± SEM of 4 independent experiments. The AXL internalization rates (dashed lines) were 8.48 ± 1.28 and 12.43 ± 0.48 %/min in MCF10AmCh and MCF10AF1F2 cells, respectively, revealing a significant 1.45-fold increase (p=0.0088) upon flotillin upregulation. Comparison of the AXL internalization rates (dashed lines) in MDA-MB-231shLuci and MDA-MB-231shFlot2 cells showed a significant 3.66-fold decrease (p=0.0009) upon flotillin downregulation. Results are expressed as the percentage of the maximum level of surface labeled AXL at T0 and are the mean ± SEM of 4 independent experiments. G) Flotillin upregulation promotes AXL protein level increase . Cell lysates from MCF10AmCh and MCF10AF1F2 cells were probed by western blotting with antibodies against AXL phosphorylated on Y702, AXL and tubulin. Results are expressed as fold-increase compared with MCF10AmCh cells and are the mean ± SEM of 6 independent experiments. H) Flotillin upregulation does not increase AXL mRNA level. RT-qPCR analysis of AXL expression in MCF10AmCh and MCF10AF1F2 cells. Results are expressed relative to the level in MCF10AmCh cells and are the mean ± SEM of 4 independent experiments. I) Flotillin upregulation stabilizes AXL. MCF10A and MCF10AF1F2 cells were incubated with cycloheximide (CHX, 100µg/ml) and cell lysates collected at the indicated time points. AXL and tubulin levels were assessed by western blotting. The graph shows the normalized AXL levels in each cell line during CHX incubation. CHX treatment was stopped after 9 hours because MCF10Amch cells did not survive longer treatment. *P

    Journal: bioRxiv

    Article Title: Flotillin-upregulation acts as an epithelial-mesenchymal transition driver by promoting sphingosine kinase 2-dependent AXL stabilization

    doi: 10.1101/2020.05.12.090571

    Figure Lengend Snippet: Flotillin upregulation stimulates AXL endocytosis towards non-degradative late endosomes and promotes its stabilization. A) AXL knock-down in MCF10AF1F2 cells decreases ZEB1 and N-cadherin expression . MCF10AmCh and MCF10AF1F2 cells were transfected with control siRNA (CTL) or against AXL and probed by western blotting with antibodies for the indicated proteins. The quantification results are the mean ± SEM of 4 independent experiment, and are expressed as fold-increase compared with control (MCF10AmCh). B) ZEB1 nuclear localization analyzed by immunocytochemistry and co-staining with Hoechst (Hst) and C) E-cadherin distribution analyzed by immunocytochemistry in MCF10AmCh and MCF10AF1F2 cells transfected with control (CTL) or anti-AXL siRNAs. Results shown are representative of 4 independent experiments. Scale bar: 10 µm. C) Flotillin-oligomerization induced by a CRY2-CIBN-based optogenetic approach promotes co-clustering of AXL . Still images from a TIRF-microscopy movie of a MCF10A cell that co-expresses CRY2-mCitrin, flotillin 2-CIBN-mCherry, and AXL-Halo labeled with Halo-Tag-Janelia Fluor 646. Representative image of a whole cell 15s after starting the 488 nm illumination to perform CRY2 binding to flotillin 2-CIBN-mCh and subsequent oligomerization (see also supplementary Fig. S6 for the details regarding the validation of the constructs and the optogenetic approach). Magnified images from the boxed area taken before and after 488 nm illumination. Arrowheads indicate clusters of CRY2-mCitrin and flotillin 2-CIBN-mCherry where AXL-Halo accumulates (see also video 4). Scale bars: 10 µm for the main image and 2 µm for the images from the boxed area. D) Co-accumulation of AXL and flotillin 2 at the PM prior to their co-endocytosis . Still images of a time-lapse series of one MCF10AF1F2 cell that expresses AXL-GFP to show flotillin 2/AXL co-localization at the PM and their co-endocytosis at PM sites (showed by arrowheads). Kymographs show the temporal variation of the GFP and mCherry signals along two line-scans (blue at the PM endocytic site, and yellow close to the PM endocytic site). Graphs show the intensity variation of both signals along the dotted white lines on the kymographs. The left kymograph shows the accumulation of AXL-GFP and flotillin 2-mCherry at the PM followed by a sudden concomitant drop of both signals (around 83s, indicated by a star). The right kymograph shows that in the meantime, both AXL-GFP and flotillin 2-mCherry signals remained stable at the PM outside and close to this endocytic site (See also video 5). E) Flotillin 1 co-precipitates with AXL. AXL was immunoprecipitated from MCF10AF1F2 cell lysates, and flotillin 1 co-immunoprecipitation was assessed by immunoblotting. Control immunoprecipitation was performed with non-relevant immunoglobulins (Ig). F) Flotillin upregulation accelerates AXL endocytosis. Kinetics of AXL internalization in MCF10AmCh and MCF10AF1F2 cells (left), and in MDA-MB-231shLuci and MDA-MB-231shFlot2 cells (right). Surface proteins were labeled with biotin at 4°C, and cells were incubated at 37°C for the indicated times to allow endocytosis. AXL presence in the internalized biotinylated fraction was probed by western blotting using relevant antibodies and quantified as the percentage of the maximum level of internalized protein (reached at 12 and 8 min, respectively, in MCF10AmCh and MCF10AF1F2 cells). Results are expressed as the mean ± SEM of 4 independent experiments. The AXL internalization rates (dashed lines) were 8.48 ± 1.28 and 12.43 ± 0.48 %/min in MCF10AmCh and MCF10AF1F2 cells, respectively, revealing a significant 1.45-fold increase (p=0.0088) upon flotillin upregulation. Comparison of the AXL internalization rates (dashed lines) in MDA-MB-231shLuci and MDA-MB-231shFlot2 cells showed a significant 3.66-fold decrease (p=0.0009) upon flotillin downregulation. Results are expressed as the percentage of the maximum level of surface labeled AXL at T0 and are the mean ± SEM of 4 independent experiments. G) Flotillin upregulation promotes AXL protein level increase . Cell lysates from MCF10AmCh and MCF10AF1F2 cells were probed by western blotting with antibodies against AXL phosphorylated on Y702, AXL and tubulin. Results are expressed as fold-increase compared with MCF10AmCh cells and are the mean ± SEM of 6 independent experiments. H) Flotillin upregulation does not increase AXL mRNA level. RT-qPCR analysis of AXL expression in MCF10AmCh and MCF10AF1F2 cells. Results are expressed relative to the level in MCF10AmCh cells and are the mean ± SEM of 4 independent experiments. I) Flotillin upregulation stabilizes AXL. MCF10A and MCF10AF1F2 cells were incubated with cycloheximide (CHX, 100µg/ml) and cell lysates collected at the indicated time points. AXL and tubulin levels were assessed by western blotting. The graph shows the normalized AXL levels in each cell line during CHX incubation. CHX treatment was stopped after 9 hours because MCF10Amch cells did not survive longer treatment. *P

    Article Snippet: Antibodies and reagents Mouse antibodies used were against: actin (A5441, Sigma), flotillin 1 and flotillin 2 (BD Biosciences), E-cadherin (used for MCF10A cells, Life-technologies), E-cadherin (used for NMuMG cells, BD Biosciences), LAMP1 (used for MCF10A cells, BD Biosciences), Smad3 (ThermoFisher), vimentin and α-tubulin (Sigma), phosphorylated ERK1/2 at T202/Y204 and AKT (CST), pan-Ras (Millipore), phosphorylated Y (4G10), EphA4 (gift from Dr Greenberg, Harvard, Boston, USA) , and STAT3 (CST, #9139S).

    Techniques: Expressing, Transfection, Western Blot, Immunocytochemistry, Staining, Microscopy, Labeling, Binding Assay, Construct, Immunoprecipitation, Multiple Displacement Amplification, Incubation, Quantitative RT-PCR

    AXL surface staining in MDA-MB-231 cells and AXL and CD71 uptake. A,B) AXL level at the cell surface is increased in MDA-MB-231 cells in which flotillins were knocked down. (A)  In fixed non-permeabilized cells, AXL expression was analyzed using the monoclonal D4 antibody against AXL extracellular domain and an Alexa488-conjugated anti-human secondary antibody. Images were acquired in the same illumination conditions for MDA-MB-shLuci cells (control) and MDA-MB-231-shFlot2cells (in which flotillins were knocked down). Using the Fiji software, the mean fluorescence intensity was quantified in each area delimited by the cellular outline. Quantification was done in 53 MDA-MB-shLuci cells and 41 MDA-MB-231-shFlot2cells. The values were  expressed as arbitrary units correspond to the mean ± SEM.  (B)  AXL surface staining analyzed by FACS. Fixed, non-permeabilized cells were labeled with the D9 anti-AXL antibody and a Alexa488-conjugated secondary antibody and analyzed by FACS. Results are expressed as the Mean Fluorescence Intensity (MFI) from 3 independent experiments.  A) (C)  AXL detection by western blotting in whole cell lysates of MDA-MB-231shLuci and shFlot2 cells. Actin was used as loading control. Results are expressed as fold-increase compared with MDA-MB-shLuci cells and are the mean ± SEM of 4 independent experiments.  B) D)  MCF10AmCh and MCF10AF1F2 cells were incubated with the anti-AXL D4 antibody (against AXL extracellular domain) at 4°C followed by incubation at 37°C for 10 min to allow AXL internalization. After fixation and permeabilization, internalized AXL was labeled (green) with a FITC-conjugated secondary antibody directed against the D4 antibody. Immunofluorescence staining of AXL (blue) was also performed using an antibody against AXL cytoplasmic domain and an Alexa-633 conjugated secondary antibody. Arrowheads indicate flotillin 2-mCherry-positive vesicles in which AXL is internalized (green). Images are representative of at least 5 independent experiments.  C) E)  MCF10AmCh and MCF10AF1F2 cells were incubated with the anti-AXL D4 antibody at 4°C followed by incubation at 37°C for 10 min to allow AXL internalization. AXL distribution and flotillin 2-mCherry signal were analyzed by immunocytochemistry. Arrowheads indicate AXL/flotillin 2 colocalization. Images are representative of at least 5 independent experiments. Scale bars are 10 µm in the main images, and 1 µm in the magnified images from the insets.  D) F)  MCF10AmCh and MCF10AF1F2 cells that express Myc-tagged AXL were incubated with an anti-Myc antibody at 4°C followed by incubation at 37°C for the indicated time. Myc-AXL distribution was analyzed by immunocytochemistry using an Alexa488-conjugated secondary antibody. Scale bars = 10 µm in the main images, and 1 µm in the magnified images from the insets.  E) G) Flotillin upregulation does not affect transferrin receptor endocytosis.  Kinetics of CD71 (transferrin receptor) internalization in MCF10AmCh and MCF10AF1F2 cells analyzed in the same experiments presented in   Figure 6  G. Surface proteins were labeled with biotin at 4°C and cells incubated at 37°C for the indicated times to allow endocytosis. CD71 presence in the internalized biotinylated fraction was probed by western blotting using relevant antibodies, and quantified as the percentage of the maximum level of internalized protein. Results are expressed as the mean ± SEM of 4 independent experiments. The CD71 internalization rates were not significantly different between cell lines at 4, 8 and 12 min, p > 0.1.  F) H) Flotillin upregulation does not affect CD71 level.  Cell lysates of MCF10AmCh and MCF10AF1F2 cells were probed by western blotting with antibodies against CD71 and actin. Results are expressed as fold-increase compared with MCF10AmCh cells and are the mean ± SEM of 4 independent experiments.

    Journal: bioRxiv

    Article Title: Flotillin-upregulation acts as an epithelial-mesenchymal transition driver by promoting sphingosine kinase 2-dependent AXL stabilization

    doi: 10.1101/2020.05.12.090571

    Figure Lengend Snippet: AXL surface staining in MDA-MB-231 cells and AXL and CD71 uptake. A,B) AXL level at the cell surface is increased in MDA-MB-231 cells in which flotillins were knocked down. (A) In fixed non-permeabilized cells, AXL expression was analyzed using the monoclonal D4 antibody against AXL extracellular domain and an Alexa488-conjugated anti-human secondary antibody. Images were acquired in the same illumination conditions for MDA-MB-shLuci cells (control) and MDA-MB-231-shFlot2cells (in which flotillins were knocked down). Using the Fiji software, the mean fluorescence intensity was quantified in each area delimited by the cellular outline. Quantification was done in 53 MDA-MB-shLuci cells and 41 MDA-MB-231-shFlot2cells. The values were expressed as arbitrary units correspond to the mean ± SEM. (B) AXL surface staining analyzed by FACS. Fixed, non-permeabilized cells were labeled with the D9 anti-AXL antibody and a Alexa488-conjugated secondary antibody and analyzed by FACS. Results are expressed as the Mean Fluorescence Intensity (MFI) from 3 independent experiments. A) (C) AXL detection by western blotting in whole cell lysates of MDA-MB-231shLuci and shFlot2 cells. Actin was used as loading control. Results are expressed as fold-increase compared with MDA-MB-shLuci cells and are the mean ± SEM of 4 independent experiments. B) D) MCF10AmCh and MCF10AF1F2 cells were incubated with the anti-AXL D4 antibody (against AXL extracellular domain) at 4°C followed by incubation at 37°C for 10 min to allow AXL internalization. After fixation and permeabilization, internalized AXL was labeled (green) with a FITC-conjugated secondary antibody directed against the D4 antibody. Immunofluorescence staining of AXL (blue) was also performed using an antibody against AXL cytoplasmic domain and an Alexa-633 conjugated secondary antibody. Arrowheads indicate flotillin 2-mCherry-positive vesicles in which AXL is internalized (green). Images are representative of at least 5 independent experiments. C) E) MCF10AmCh and MCF10AF1F2 cells were incubated with the anti-AXL D4 antibody at 4°C followed by incubation at 37°C for 10 min to allow AXL internalization. AXL distribution and flotillin 2-mCherry signal were analyzed by immunocytochemistry. Arrowheads indicate AXL/flotillin 2 colocalization. Images are representative of at least 5 independent experiments. Scale bars are 10 µm in the main images, and 1 µm in the magnified images from the insets. D) F) MCF10AmCh and MCF10AF1F2 cells that express Myc-tagged AXL were incubated with an anti-Myc antibody at 4°C followed by incubation at 37°C for the indicated time. Myc-AXL distribution was analyzed by immunocytochemistry using an Alexa488-conjugated secondary antibody. Scale bars = 10 µm in the main images, and 1 µm in the magnified images from the insets. E) G) Flotillin upregulation does not affect transferrin receptor endocytosis. Kinetics of CD71 (transferrin receptor) internalization in MCF10AmCh and MCF10AF1F2 cells analyzed in the same experiments presented in Figure 6 G. Surface proteins were labeled with biotin at 4°C and cells incubated at 37°C for the indicated times to allow endocytosis. CD71 presence in the internalized biotinylated fraction was probed by western blotting using relevant antibodies, and quantified as the percentage of the maximum level of internalized protein. Results are expressed as the mean ± SEM of 4 independent experiments. The CD71 internalization rates were not significantly different between cell lines at 4, 8 and 12 min, p > 0.1. F) H) Flotillin upregulation does not affect CD71 level. Cell lysates of MCF10AmCh and MCF10AF1F2 cells were probed by western blotting with antibodies against CD71 and actin. Results are expressed as fold-increase compared with MCF10AmCh cells and are the mean ± SEM of 4 independent experiments.

    Article Snippet: Antibodies and reagents Mouse antibodies used were against: actin (A5441, Sigma), flotillin 1 and flotillin 2 (BD Biosciences), E-cadherin (used for MCF10A cells, Life-technologies), E-cadherin (used for NMuMG cells, BD Biosciences), LAMP1 (used for MCF10A cells, BD Biosciences), Smad3 (ThermoFisher), vimentin and α-tubulin (Sigma), phosphorylated ERK1/2 at T202/Y204 and AKT (CST), pan-Ras (Millipore), phosphorylated Y (4G10), EphA4 (gift from Dr Greenberg, Harvard, Boston, USA) , and STAT3 (CST, #9139S).

    Techniques: Staining, Multiple Displacement Amplification, Expressing, Software, Fluorescence, FACS, Labeling, Western Blot, Incubation, Immunofluorescence, Immunocytochemistry

    Flotillin upregulation promotes activation of oncogenic signaling pathways A) Human phospho-kinase arrays were incubated with MCF10AmCh and MCF10AF1F2 cell lysates as described by the manufacturer. One representative example of arrays is shown. The table lists the twelve kinases and kinase substrates with the highest ratio of phosphorylation change between MCF10AF1F2 and MCF10AmCh cells. Values are the mean of 2 independent experiments. B) The level of active GTP-bound Ras was measured using GST-Raf-RBD in lysates obtained from MCF10AmCh and MCF10AF1F2 cells. The histogram shows the mean value (± SEM) of Ras activity normalized to the amount of total protein calculated from 5 independent experiments. C, E, F, G) Cell lysates from MCF10AmCh and MCF10AF1F2 cells were analyzed by western blotting to assess the phosphorylation status of the signaling proteins ERK1/2 (C, n=4), AKT (E), STAT3 (F), and Smad3 (G), with the indicated antibodies. The histograms show the mean value (± SEM) of protein phosphorylation normalized to the total amount of the considered protein in MCF10AF1F2 cells compared with control MCF10AmCh cells calculated from 4 independent experiments. A) D) MCF10AmCh and MCF10AF1F2 cells were transfected with an ERK2-GFP plasmid and ERK2-GFP distribution was analyzed by confocal microscopy at 36 hours post-transfection. B) H) Confocal microscopy images of MCF10AmCh and MCF10AF1F2 cells incubated with an antibody against Smad3 phosphorylated on S423/425 and Hoechst (Hst) staining for nuclei. In D and H, scale bars: 10µm. *P

    Journal: bioRxiv

    Article Title: Flotillin-upregulation acts as an epithelial-mesenchymal transition driver by promoting sphingosine kinase 2-dependent AXL stabilization

    doi: 10.1101/2020.05.12.090571

    Figure Lengend Snippet: Flotillin upregulation promotes activation of oncogenic signaling pathways A) Human phospho-kinase arrays were incubated with MCF10AmCh and MCF10AF1F2 cell lysates as described by the manufacturer. One representative example of arrays is shown. The table lists the twelve kinases and kinase substrates with the highest ratio of phosphorylation change between MCF10AF1F2 and MCF10AmCh cells. Values are the mean of 2 independent experiments. B) The level of active GTP-bound Ras was measured using GST-Raf-RBD in lysates obtained from MCF10AmCh and MCF10AF1F2 cells. The histogram shows the mean value (± SEM) of Ras activity normalized to the amount of total protein calculated from 5 independent experiments. C, E, F, G) Cell lysates from MCF10AmCh and MCF10AF1F2 cells were analyzed by western blotting to assess the phosphorylation status of the signaling proteins ERK1/2 (C, n=4), AKT (E), STAT3 (F), and Smad3 (G), with the indicated antibodies. The histograms show the mean value (± SEM) of protein phosphorylation normalized to the total amount of the considered protein in MCF10AF1F2 cells compared with control MCF10AmCh cells calculated from 4 independent experiments. A) D) MCF10AmCh and MCF10AF1F2 cells were transfected with an ERK2-GFP plasmid and ERK2-GFP distribution was analyzed by confocal microscopy at 36 hours post-transfection. B) H) Confocal microscopy images of MCF10AmCh and MCF10AF1F2 cells incubated with an antibody against Smad3 phosphorylated on S423/425 and Hoechst (Hst) staining for nuclei. In D and H, scale bars: 10µm. *P

    Article Snippet: Antibodies and reagents Mouse antibodies used were against: actin (A5441, Sigma), flotillin 1 and flotillin 2 (BD Biosciences), E-cadherin (used for MCF10A cells, Life-technologies), E-cadherin (used for NMuMG cells, BD Biosciences), LAMP1 (used for MCF10A cells, BD Biosciences), Smad3 (ThermoFisher), vimentin and α-tubulin (Sigma), phosphorylated ERK1/2 at T202/Y204 and AKT (CST), pan-Ras (Millipore), phosphorylated Y (4G10), EphA4 (gift from Dr Greenberg, Harvard, Boston, USA) , and STAT3 (CST, #9139S).

    Techniques: Activation Assay, Incubation, Activity Assay, Western Blot, Transfection, Plasmid Preparation, Confocal Microscopy, Staining

    AXL, EphA4 and TGFβ receptor, but not CD71 relocate to flotillin-positive late endosomes in mammary epithelial cells upon flotillin upregulation. A)  In MCF10AF1F2 cells, endogenous AXL is localized in flotillin 2-mCherry vesicles positive for the endolysosomal marker LAMP1. Confocal microscopy image representative of several cells from independent immunofluorescence experiments.  B)  Myc-tagged AXL expressed in MCF10AF1F2 cells is localized in flotillin 2-mCherry vesicles positive for the late endosomal marker CD63. Confocal microscopy image representative of several cells from independent immunofluorescence experiments.  C)  MDA-MB-231 cells were transfected with AXL-GFP, flotillin 2-mCherry and flotillin 1-HA-encoding plasmids. Live cells were imaged by spinning disk confocal microscopy, showing the co-localization (indicated by white arrows) and the co-trafficking of AXL-GFP and flotillin 2-mCherry in intracellular vesicles (See also video 2).  D)  Still images of a spinning disk confocal video of a MCF10AF1F2 cell that expresses EphA4-GFP showing the co-localization of EphA4-GFP and flotillin 2-mCherry. Images shown are representative of several cells observed in independent experiments.  E)  MCF10AF1F2 cells that co-express the TGFβ-RI (GFP tagged) and TGFβ-RII (Flag tagged) subunits were analyzed by immunofluorescence using an anti-Flag antibody and imaged by confocal microscopy. Both subunits were concomitantly found at the PM and in intracellular vesicles (white arrows) where they colocalized with flotillin 2-mCherry. The image shown is representative of 3 independent experiments. Scale bars: 10 µm for all images, and 2 µm for the magnified images from the boxed area.  F)  Spinning disk confocal still images of one MCF10AF1F2 cell that co-expresses TGFβ-RI-GFP and TGFβ-RII-Flag, showing the co-localization of TGFβ-RI-GFP and flotillin 2-mCherry in intracellular vesicles. The different arrows follow three distinct vesicles over time. Scale bars: 10 µm for all images, and 2 µm for the magnified images from the boxed area.  G)  Transferrin receptor is not detected in flotillin-positive late endosomes. MCF10AF1F2 cells that express CD71-GFP were imaged by confocal microscopy. The image shown is representative of several cells in independent experiments. Scale bars: 10 µm in the main image and 2 µm in magnified images from the boxed area.

    Journal: bioRxiv

    Article Title: Flotillin-upregulation acts as an epithelial-mesenchymal transition driver by promoting sphingosine kinase 2-dependent AXL stabilization

    doi: 10.1101/2020.05.12.090571

    Figure Lengend Snippet: AXL, EphA4 and TGFβ receptor, but not CD71 relocate to flotillin-positive late endosomes in mammary epithelial cells upon flotillin upregulation. A) In MCF10AF1F2 cells, endogenous AXL is localized in flotillin 2-mCherry vesicles positive for the endolysosomal marker LAMP1. Confocal microscopy image representative of several cells from independent immunofluorescence experiments. B) Myc-tagged AXL expressed in MCF10AF1F2 cells is localized in flotillin 2-mCherry vesicles positive for the late endosomal marker CD63. Confocal microscopy image representative of several cells from independent immunofluorescence experiments. C) MDA-MB-231 cells were transfected with AXL-GFP, flotillin 2-mCherry and flotillin 1-HA-encoding plasmids. Live cells were imaged by spinning disk confocal microscopy, showing the co-localization (indicated by white arrows) and the co-trafficking of AXL-GFP and flotillin 2-mCherry in intracellular vesicles (See also video 2). D) Still images of a spinning disk confocal video of a MCF10AF1F2 cell that expresses EphA4-GFP showing the co-localization of EphA4-GFP and flotillin 2-mCherry. Images shown are representative of several cells observed in independent experiments. E) MCF10AF1F2 cells that co-express the TGFβ-RI (GFP tagged) and TGFβ-RII (Flag tagged) subunits were analyzed by immunofluorescence using an anti-Flag antibody and imaged by confocal microscopy. Both subunits were concomitantly found at the PM and in intracellular vesicles (white arrows) where they colocalized with flotillin 2-mCherry. The image shown is representative of 3 independent experiments. Scale bars: 10 µm for all images, and 2 µm for the magnified images from the boxed area. F) Spinning disk confocal still images of one MCF10AF1F2 cell that co-expresses TGFβ-RI-GFP and TGFβ-RII-Flag, showing the co-localization of TGFβ-RI-GFP and flotillin 2-mCherry in intracellular vesicles. The different arrows follow three distinct vesicles over time. Scale bars: 10 µm for all images, and 2 µm for the magnified images from the boxed area. G) Transferrin receptor is not detected in flotillin-positive late endosomes. MCF10AF1F2 cells that express CD71-GFP were imaged by confocal microscopy. The image shown is representative of several cells in independent experiments. Scale bars: 10 µm in the main image and 2 µm in magnified images from the boxed area.

    Article Snippet: Antibodies and reagents Mouse antibodies used were against: actin (A5441, Sigma), flotillin 1 and flotillin 2 (BD Biosciences), E-cadherin (used for MCF10A cells, Life-technologies), E-cadherin (used for NMuMG cells, BD Biosciences), LAMP1 (used for MCF10A cells, BD Biosciences), Smad3 (ThermoFisher), vimentin and α-tubulin (Sigma), phosphorylated ERK1/2 at T202/Y204 and AKT (CST), pan-Ras (Millipore), phosphorylated Y (4G10), EphA4 (gift from Dr Greenberg, Harvard, Boston, USA) , and STAT3 (CST, #9139S).

    Techniques: Marker, Confocal Microscopy, Immunofluorescence, Multiple Displacement Amplification, Transfection

    Model Co-upregulation of flotillin 1 and 2 favors their oligomerization and the formation of flotillin-rich microdomains at the plasma membrane. At these membrane sites, signaling receptors, particularly the tyrosine kinase receptor AXL, are trapped. Moreover, sphingosine might be concentrated in these flotillin-rich microdomains through binding to flotillins. This promotes the recruitment of SphK2 that phosphorylates sphingosine to promote the endocytosis of AXL and other receptors in their active form. Through the UFIT pathway these receptors are delivered to “flotillin-positive late endosomes” where AXL is protected from degradation, allowing its stabilization. SphK2 is abundant in these vesicular compartments and might participate in defining their “stabilizing properties”. Consequently, these flotillin-positive late endosomes act as signaling endosomes favoring the hyperactivation of oncogenic signaling pathways that promote EMT.

    Journal: bioRxiv

    Article Title: Flotillin-upregulation acts as an epithelial-mesenchymal transition driver by promoting sphingosine kinase 2-dependent AXL stabilization

    doi: 10.1101/2020.05.12.090571

    Figure Lengend Snippet: Model Co-upregulation of flotillin 1 and 2 favors their oligomerization and the formation of flotillin-rich microdomains at the plasma membrane. At these membrane sites, signaling receptors, particularly the tyrosine kinase receptor AXL, are trapped. Moreover, sphingosine might be concentrated in these flotillin-rich microdomains through binding to flotillins. This promotes the recruitment of SphK2 that phosphorylates sphingosine to promote the endocytosis of AXL and other receptors in their active form. Through the UFIT pathway these receptors are delivered to “flotillin-positive late endosomes” where AXL is protected from degradation, allowing its stabilization. SphK2 is abundant in these vesicular compartments and might participate in defining their “stabilizing properties”. Consequently, these flotillin-positive late endosomes act as signaling endosomes favoring the hyperactivation of oncogenic signaling pathways that promote EMT.

    Article Snippet: Antibodies and reagents Mouse antibodies used were against: actin (A5441, Sigma), flotillin 1 and flotillin 2 (BD Biosciences), E-cadherin (used for MCF10A cells, Life-technologies), E-cadherin (used for NMuMG cells, BD Biosciences), LAMP1 (used for MCF10A cells, BD Biosciences), Smad3 (ThermoFisher), vimentin and α-tubulin (Sigma), phosphorylated ERK1/2 at T202/Y204 and AKT (CST), pan-Ras (Millipore), phosphorylated Y (4G10), EphA4 (gift from Dr Greenberg, Harvard, Boston, USA) , and STAT3 (CST, #9139S).

    Techniques: Binding Assay

    Flotillin upregulation in non-tumoral epithelial mammary MCF10A cells induces EMT. A) GO identification of the biological process, cellular component and molecular function terms associated with the 802 genes that were differentially expressed in MCF10AF1F2 cells compared with MCF10AmCh cells. B) Venn diagrams comparing the genes that we found to be up- or down-regulated ( > or

    Journal: bioRxiv

    Article Title: Flotillin-upregulation acts as an epithelial-mesenchymal transition driver by promoting sphingosine kinase 2-dependent AXL stabilization

    doi: 10.1101/2020.05.12.090571

    Figure Lengend Snippet: Flotillin upregulation in non-tumoral epithelial mammary MCF10A cells induces EMT. A) GO identification of the biological process, cellular component and molecular function terms associated with the 802 genes that were differentially expressed in MCF10AF1F2 cells compared with MCF10AmCh cells. B) Venn diagrams comparing the genes that we found to be up- or down-regulated ( > or

    Article Snippet: Antibodies and reagents Mouse antibodies used were against: actin (A5441, Sigma), flotillin 1 and flotillin 2 (BD Biosciences), E-cadherin (used for MCF10A cells, Life-technologies), E-cadherin (used for NMuMG cells, BD Biosciences), LAMP1 (used for MCF10A cells, BD Biosciences), Smad3 (ThermoFisher), vimentin and α-tubulin (Sigma), phosphorylated ERK1/2 at T202/Y204 and AKT (CST), pan-Ras (Millipore), phosphorylated Y (4G10), EphA4 (gift from Dr Greenberg, Harvard, Boston, USA) , and STAT3 (CST, #9139S).

    Techniques:

    N-Ras and H-Ras distribution is not affected by flotillin upregulation. Confocal images of MCF10AmCh and MCF10AF1F2 cells that express H-Ras-GFP or N-Ras-GFP. The distribution of these isoforms was not altered by flotillin upregulation. H-Ras-GFP was occasionally found in flotillin 2-mCherry-positive vesicles, but this was very rare for N-Ras-GFP. Images are representative of 3 independent experiments. Scale bars: 10 µm in the main images, and 2 µm in the magnified images from the boxed area.

    Journal: bioRxiv

    Article Title: Flotillin-upregulation acts as an epithelial-mesenchymal transition driver by promoting sphingosine kinase 2-dependent AXL stabilization

    doi: 10.1101/2020.05.12.090571

    Figure Lengend Snippet: N-Ras and H-Ras distribution is not affected by flotillin upregulation. Confocal images of MCF10AmCh and MCF10AF1F2 cells that express H-Ras-GFP or N-Ras-GFP. The distribution of these isoforms was not altered by flotillin upregulation. H-Ras-GFP was occasionally found in flotillin 2-mCherry-positive vesicles, but this was very rare for N-Ras-GFP. Images are representative of 3 independent experiments. Scale bars: 10 µm in the main images, and 2 µm in the magnified images from the boxed area.

    Article Snippet: Antibodies and reagents Mouse antibodies used were against: actin (A5441, Sigma), flotillin 1 and flotillin 2 (BD Biosciences), E-cadherin (used for MCF10A cells, Life-technologies), E-cadherin (used for NMuMG cells, BD Biosciences), LAMP1 (used for MCF10A cells, BD Biosciences), Smad3 (ThermoFisher), vimentin and α-tubulin (Sigma), phosphorylated ERK1/2 at T202/Y204 and AKT (CST), pan-Ras (Millipore), phosphorylated Y (4G10), EphA4 (gift from Dr Greenberg, Harvard, Boston, USA) , and STAT3 (CST, #9139S).

    Techniques:

    Flotillin 2 is upregulated in vivo in neural crest cells (NCC) undergoing EMT in zebrafish embryos, and is required for their migration and the formation of NCC tissue derivatives. A) Endogenous flotillin expression in neural crest cells. Representative confocal images of endogenous flotillin 2 overexpression (red) in migrating cranial NCC (green) in Tg( foxd3:GFP ) embryos at 18hpf. Scale bar: 10µm. B-D) Flotillin2 is necessary for neural crest migration. B: Control (MoCTL) or flotillin 2 morpholinos (MoFlot2) were injected in Tg( foxd3:GFP ) embryos at the 1-cell stage. At 18hpf, embryos were mounted for time-lapse microscopy to analyze NCC migration. (C): Representative images of NCC in control (MoCTL) and MoFlot2 embryos. Some NCCs are colored to follow their movement during the recording period. Scale bar: 20µm. Representative NCC trajectories in the X or Y axis during 2 hours. A) (D) Boxplots showing the quantification of the velocity and persistence of migration in 8hpf control (MoCTL) and MoFlot2 embryos for 2 hours. At least 15 embryos were analyzed, and 50 cells were tracked for each condition. The values of velocity and persistence are the mean + SEM of 3 independent experiments, * p

    Journal: bioRxiv

    Article Title: Flotillin-upregulation acts as an epithelial-mesenchymal transition driver by promoting sphingosine kinase 2-dependent AXL stabilization

    doi: 10.1101/2020.05.12.090571

    Figure Lengend Snippet: Flotillin 2 is upregulated in vivo in neural crest cells (NCC) undergoing EMT in zebrafish embryos, and is required for their migration and the formation of NCC tissue derivatives. A) Endogenous flotillin expression in neural crest cells. Representative confocal images of endogenous flotillin 2 overexpression (red) in migrating cranial NCC (green) in Tg( foxd3:GFP ) embryos at 18hpf. Scale bar: 10µm. B-D) Flotillin2 is necessary for neural crest migration. B: Control (MoCTL) or flotillin 2 morpholinos (MoFlot2) were injected in Tg( foxd3:GFP ) embryos at the 1-cell stage. At 18hpf, embryos were mounted for time-lapse microscopy to analyze NCC migration. (C): Representative images of NCC in control (MoCTL) and MoFlot2 embryos. Some NCCs are colored to follow their movement during the recording period. Scale bar: 20µm. Representative NCC trajectories in the X or Y axis during 2 hours. A) (D) Boxplots showing the quantification of the velocity and persistence of migration in 8hpf control (MoCTL) and MoFlot2 embryos for 2 hours. At least 15 embryos were analyzed, and 50 cells were tracked for each condition. The values of velocity and persistence are the mean + SEM of 3 independent experiments, * p

    Article Snippet: Antibodies and reagents Mouse antibodies used were against: actin (A5441, Sigma), flotillin 1 and flotillin 2 (BD Biosciences), E-cadherin (used for MCF10A cells, Life-technologies), E-cadherin (used for NMuMG cells, BD Biosciences), LAMP1 (used for MCF10A cells, BD Biosciences), Smad3 (ThermoFisher), vimentin and α-tubulin (Sigma), phosphorylated ERK1/2 at T202/Y204 and AKT (CST), pan-Ras (Millipore), phosphorylated Y (4G10), EphA4 (gift from Dr Greenberg, Harvard, Boston, USA) , and STAT3 (CST, #9139S).

    Techniques: In Vivo, Migration, Expressing, Over Expression, Injection, Time-lapse Microscopy

    SphK1 and 2 localization and effect of SphK inhibitors. A) SphK1 is barely detected in flotillin-positive endosomes.  Live MCF10AF1F2 cells that express SphK1-GFP (A), and MDA-MB-231 cells that co-express flotillin 1-mCherry and SphK1-GFP (B) were imaged by spinning disk confocal microscopy (see quantification in   Fig. 7  B).  B, C) SphK2 colocalizes with flotillins in Rab7 and CD63 positive vesicles.  Still images of live MCF10AF1F2 cells that express SphK2-Halo labeled with Halo-tag-Janelia Fluor 646 and Rab7-GFP (B), or CD63-GFP (C) imaged by spinning disk confocal microscopy. 1.  D) Flotillin level does not modulate SphK2 protein expression.  SphK2 and tubulin expression were assessed in whole cell extracts from MCF10AmCh, MCF10AF1F2, MDA-MB-231shLuci, and MDA-MB-231shFlot2 cells by western blotting using specific antibodies. The histograms show SphK2 level (normalized to tubulin) expressed as fold-increase compared with MCF10AmCh or MDA-MB-231shLuci cells, and data are the mean ± SEM of 4 independent experiments. 2.  E) SphK2 localization in CD63-positive endosomes is increased in MCF10AF1F2 cells.  MCF10AmCh and MCF10A F1F2 cells were co-transfected with CD63-GFP and SphK2-Halo labeled with Halo-Tag-Janelia Fluor 646. Representative images acquired by spinning disk confocal microscopy on live cells are shown. To better visualize the co-localization of CD63-GFP and SphK2-Halo, the SphK2-Halo and flotillin 2-mCherry signals were pseudocolored in red and blue, respectively. The presence of SphK2-Halo in CD63-GFP-positive vesicles was quantified (n=372 vesicles in 10 MCF10AmCh cells, and n=455 vesicles in 10 MCF10AF1F2 cells). Results are expressed as the percentage per cell of CD63-GFP labelled vesicles positive for SphK2-Halo. Scale bar: 10 µm. ** p

    Journal: bioRxiv

    Article Title: Flotillin-upregulation acts as an epithelial-mesenchymal transition driver by promoting sphingosine kinase 2-dependent AXL stabilization

    doi: 10.1101/2020.05.12.090571

    Figure Lengend Snippet: SphK1 and 2 localization and effect of SphK inhibitors. A) SphK1 is barely detected in flotillin-positive endosomes. Live MCF10AF1F2 cells that express SphK1-GFP (A), and MDA-MB-231 cells that co-express flotillin 1-mCherry and SphK1-GFP (B) were imaged by spinning disk confocal microscopy (see quantification in Fig. 7 B). B, C) SphK2 colocalizes with flotillins in Rab7 and CD63 positive vesicles. Still images of live MCF10AF1F2 cells that express SphK2-Halo labeled with Halo-tag-Janelia Fluor 646 and Rab7-GFP (B), or CD63-GFP (C) imaged by spinning disk confocal microscopy. 1. D) Flotillin level does not modulate SphK2 protein expression. SphK2 and tubulin expression were assessed in whole cell extracts from MCF10AmCh, MCF10AF1F2, MDA-MB-231shLuci, and MDA-MB-231shFlot2 cells by western blotting using specific antibodies. The histograms show SphK2 level (normalized to tubulin) expressed as fold-increase compared with MCF10AmCh or MDA-MB-231shLuci cells, and data are the mean ± SEM of 4 independent experiments. 2. E) SphK2 localization in CD63-positive endosomes is increased in MCF10AF1F2 cells. MCF10AmCh and MCF10A F1F2 cells were co-transfected with CD63-GFP and SphK2-Halo labeled with Halo-Tag-Janelia Fluor 646. Representative images acquired by spinning disk confocal microscopy on live cells are shown. To better visualize the co-localization of CD63-GFP and SphK2-Halo, the SphK2-Halo and flotillin 2-mCherry signals were pseudocolored in red and blue, respectively. The presence of SphK2-Halo in CD63-GFP-positive vesicles was quantified (n=372 vesicles in 10 MCF10AmCh cells, and n=455 vesicles in 10 MCF10AF1F2 cells). Results are expressed as the percentage per cell of CD63-GFP labelled vesicles positive for SphK2-Halo. Scale bar: 10 µm. ** p

    Article Snippet: Antibodies and reagents Mouse antibodies used were against: actin (A5441, Sigma), flotillin 1 and flotillin 2 (BD Biosciences), E-cadherin (used for MCF10A cells, Life-technologies), E-cadherin (used for NMuMG cells, BD Biosciences), LAMP1 (used for MCF10A cells, BD Biosciences), Smad3 (ThermoFisher), vimentin and α-tubulin (Sigma), phosphorylated ERK1/2 at T202/Y204 and AKT (CST), pan-Ras (Millipore), phosphorylated Y (4G10), EphA4 (gift from Dr Greenberg, Harvard, Boston, USA) , and STAT3 (CST, #9139S).

    Techniques: Multiple Displacement Amplification, Confocal Microscopy, Labeling, Expressing, Western Blot, Transfection

    Flotillin-1 knock-down does not affect mRNA expression of caveolin-1. SK-CO15 cells were transfected with flotillin-1 or control siRNAs and 72 h post-transfection were processed for RNA isolation and real-time RT PCR quantification of caveolin-1 mRNA expression. Individual amplification curves for caveolin-1 and a housekeeping control, 18S rRNA (A) and calculated threshold cycle number for caveolin-1 amplification (B) are shown. Note identical threshold cycle number for caveolin-1 amplification in flotillin-1-depleted and control SK-CO15 cells. Data presented as means ± SE ( n = 3).

    Journal: Biochemical and biophysical research communications

    Article Title: Flotillin-1 stabilizes caveolin-1 in intestinal epithelial cells

    doi: 10.1016/j.bbrc.2008.12.118

    Figure Lengend Snippet: Flotillin-1 knock-down does not affect mRNA expression of caveolin-1. SK-CO15 cells were transfected with flotillin-1 or control siRNAs and 72 h post-transfection were processed for RNA isolation and real-time RT PCR quantification of caveolin-1 mRNA expression. Individual amplification curves for caveolin-1 and a housekeeping control, 18S rRNA (A) and calculated threshold cycle number for caveolin-1 amplification (B) are shown. Note identical threshold cycle number for caveolin-1 amplification in flotillin-1-depleted and control SK-CO15 cells. Data presented as means ± SE ( n = 3).

    Article Snippet: The following primary polyclonal (pAb) and monoclonal (mAb) antibodies were used for immunoblotting: flotillin-1 mAb clone 18, flotillin-2 mAb clone 29, caveolin- 1 pAb (BD Biosciences, San Jose, CA); α-actin pAb (Sigma–Aldrich, St. Louis, MO).

    Techniques: Expressing, Transfection, Isolation, Quantitative RT-PCR, Amplification

    Down-regulation of flotillin-1 and flotillin-2 expression inhibits lipid raft-mediated endocytosis. SK-CO15 cells were transfected with flotillin-1, flotillin-2 or control siRNAs and were subjected to the BODIPY-LacCer internalization assay 72 h post-transfection. Representative images of internalized LacCer (green, A) and pixel intensity quantification of internalized dye fluorescence (B) are shown. Note significant decrease in intracellular level of BODIPY-LacCer in flotillin-1 and flotillin-2 depleted cells. Data presented as means ± SE ( n = 14), * P

    Journal: Biochemical and biophysical research communications

    Article Title: Flotillin-1 stabilizes caveolin-1 in intestinal epithelial cells

    doi: 10.1016/j.bbrc.2008.12.118

    Figure Lengend Snippet: Down-regulation of flotillin-1 and flotillin-2 expression inhibits lipid raft-mediated endocytosis. SK-CO15 cells were transfected with flotillin-1, flotillin-2 or control siRNAs and were subjected to the BODIPY-LacCer internalization assay 72 h post-transfection. Representative images of internalized LacCer (green, A) and pixel intensity quantification of internalized dye fluorescence (B) are shown. Note significant decrease in intracellular level of BODIPY-LacCer in flotillin-1 and flotillin-2 depleted cells. Data presented as means ± SE ( n = 14), * P

    Article Snippet: The following primary polyclonal (pAb) and monoclonal (mAb) antibodies were used for immunoblotting: flotillin-1 mAb clone 18, flotillin-2 mAb clone 29, caveolin- 1 pAb (BD Biosciences, San Jose, CA); α-actin pAb (Sigma–Aldrich, St. Louis, MO).

    Techniques: Expressing, Transfection, Fluorescence

    Down-regulation of flotillin-1 expression decreases caveolin-1 level in intestinal epithelial cells. SK-CO15 cells were transfected with flotillin-1, flotillin-2, caveolin-1 or control siRNAs and expression of lipid raft-resident proteins in these cells was analyzed by immunoblotting 72 h post-transfection. Representative immunoblots (A and C) and densitometric quantification of immunoreactive bands (B) are shown. Note dramatic down-regulation of caveolin-1 expression in flotillin-1 but not flotillin-2-depleted cells and lack of effect of caveolin-1 knock-down on flotillin isoforms level. Data presented as means ± SE ( n = 5), * P

    Journal: Biochemical and biophysical research communications

    Article Title: Flotillin-1 stabilizes caveolin-1 in intestinal epithelial cells

    doi: 10.1016/j.bbrc.2008.12.118

    Figure Lengend Snippet: Down-regulation of flotillin-1 expression decreases caveolin-1 level in intestinal epithelial cells. SK-CO15 cells were transfected with flotillin-1, flotillin-2, caveolin-1 or control siRNAs and expression of lipid raft-resident proteins in these cells was analyzed by immunoblotting 72 h post-transfection. Representative immunoblots (A and C) and densitometric quantification of immunoreactive bands (B) are shown. Note dramatic down-regulation of caveolin-1 expression in flotillin-1 but not flotillin-2-depleted cells and lack of effect of caveolin-1 knock-down on flotillin isoforms level. Data presented as means ± SE ( n = 5), * P

    Article Snippet: The following primary polyclonal (pAb) and monoclonal (mAb) antibodies were used for immunoblotting: flotillin-1 mAb clone 18, flotillin-2 mAb clone 29, caveolin- 1 pAb (BD Biosciences, San Jose, CA); α-actin pAb (Sigma–Aldrich, St. Louis, MO).

    Techniques: Expressing, Transfection, Western Blot

    Flotillin-1 knock-down decreases protein level of caveolin-1 by accelerating its lysosomal degradation in cholesterol-independent fashion. SK-CO15 cells were transfected with flotillin-1 or control siRNAs and 50 h post-transfection exposed to either lysosomal inhibitor chloroquine (CQ, 0.1 mM), proteosomal inhibitor MG262 (10 nm) or vehicle for additional 22 h. Expression of resident lipid raft proteins was analyzed by immunoblotting. Representative immunoblots (A) and densitometric quantification of immunoreactive bands (B) are shown. Note restoration of caveolin-1 expression in flotillin-1-depleted cells treated by chloroquine but not MG262. Data presented as means ± SE ( n = 3), * P

    Journal: Biochemical and biophysical research communications

    Article Title: Flotillin-1 stabilizes caveolin-1 in intestinal epithelial cells

    doi: 10.1016/j.bbrc.2008.12.118

    Figure Lengend Snippet: Flotillin-1 knock-down decreases protein level of caveolin-1 by accelerating its lysosomal degradation in cholesterol-independent fashion. SK-CO15 cells were transfected with flotillin-1 or control siRNAs and 50 h post-transfection exposed to either lysosomal inhibitor chloroquine (CQ, 0.1 mM), proteosomal inhibitor MG262 (10 nm) or vehicle for additional 22 h. Expression of resident lipid raft proteins was analyzed by immunoblotting. Representative immunoblots (A) and densitometric quantification of immunoreactive bands (B) are shown. Note restoration of caveolin-1 expression in flotillin-1-depleted cells treated by chloroquine but not MG262. Data presented as means ± SE ( n = 3), * P

    Article Snippet: The following primary polyclonal (pAb) and monoclonal (mAb) antibodies were used for immunoblotting: flotillin-1 mAb clone 18, flotillin-2 mAb clone 29, caveolin- 1 pAb (BD Biosciences, San Jose, CA); α-actin pAb (Sigma–Aldrich, St. Louis, MO).

    Techniques: Transfection, Expressing, Western Blot

    Knockdown of flotillin-1 or flotillin-2 results in altered focal adhesion morphology during cell spreading. HeLa cells were transfected with the indicated siRNAs. The cells were detached, kept in suspension, and then seeded on fibronectin for 25 min. Focal adhesions were visualized by vinculin staining and filamentous actin by phalloidin staining. Scale bar: 10 µm, the same magnification for all images.

    Journal: Cells

    Article Title: Flotillins Regulate Focal Adhesions by Interacting with α-Actinin and by Influencing the Activation of Focal Adhesion Kinase

    doi: 10.3390/cells7040028

    Figure Lengend Snippet: Knockdown of flotillin-1 or flotillin-2 results in altered focal adhesion morphology during cell spreading. HeLa cells were transfected with the indicated siRNAs. The cells were detached, kept in suspension, and then seeded on fibronectin for 25 min. Focal adhesions were visualized by vinculin staining and filamentous actin by phalloidin staining. Scale bar: 10 µm, the same magnification for all images.

    Article Snippet: Antibodies Flotillin-1 and flotillin-2: mouse monoclonal (BD Biosciences, Heidelberg, Germany) and rabbit polyclonal (Sigma-Aldrich) antibodies; vinculin: mouse monoclonal antibody (Sigma-Aldrich); FAK and pY397-FAK: mouse monoclonal (BD Biosciences); ERK1/2: rabbit polyclonal (C-14, Santa Cruz Biotechnology, Heidelberg, Germany) antibodies; pERK2: mouse monoclonal antibody (E-4, Santa Cruz); GAPDH: mouse monoclonal antibody (Abcam, Cambridge, UK); α-actinin: rabbit polyclonal H-300 antibody (Santa Cruz) for IP, mouse monoclonal antibody (BD Biosciences) for Western blot and mouse monoclonal antibody (BM-75-1, Sigma-Aldrich) for immunofluorescence; His-tag: mouse monoclonal antibody (Novagen, Darmstadt, Germany), myc-tag: mouse monoclonal antibody (Santa Cruz Biotechnology).

    Techniques: Transfection, Staining

    Knockdown of flotillins results in altered morphology and length of focal adhesions during cell spreading. HeLa cells were transfected with control siRNAs or siRNAs against flotillin-1 or flotillin-2. ( a ) The cells were detached, seeded for 25 min on fibronectin, and stained for α-actinin and FAK. (Scale bars = 10 µm) ( b ) The quantification of FA length was performed with Image J using FAK staining. The graph shows the length of FAs in nm. For the quantification, at least nine cells (60–180 FAs) from three individual experiments were quantified. (** p

    Journal: Cells

    Article Title: Flotillins Regulate Focal Adhesions by Interacting with α-Actinin and by Influencing the Activation of Focal Adhesion Kinase

    doi: 10.3390/cells7040028

    Figure Lengend Snippet: Knockdown of flotillins results in altered morphology and length of focal adhesions during cell spreading. HeLa cells were transfected with control siRNAs or siRNAs against flotillin-1 or flotillin-2. ( a ) The cells were detached, seeded for 25 min on fibronectin, and stained for α-actinin and FAK. (Scale bars = 10 µm) ( b ) The quantification of FA length was performed with Image J using FAK staining. The graph shows the length of FAs in nm. For the quantification, at least nine cells (60–180 FAs) from three individual experiments were quantified. (** p

    Article Snippet: Antibodies Flotillin-1 and flotillin-2: mouse monoclonal (BD Biosciences, Heidelberg, Germany) and rabbit polyclonal (Sigma-Aldrich) antibodies; vinculin: mouse monoclonal antibody (Sigma-Aldrich); FAK and pY397-FAK: mouse monoclonal (BD Biosciences); ERK1/2: rabbit polyclonal (C-14, Santa Cruz Biotechnology, Heidelberg, Germany) antibodies; pERK2: mouse monoclonal antibody (E-4, Santa Cruz); GAPDH: mouse monoclonal antibody (Abcam, Cambridge, UK); α-actinin: rabbit polyclonal H-300 antibody (Santa Cruz) for IP, mouse monoclonal antibody (BD Biosciences) for Western blot and mouse monoclonal antibody (BM-75-1, Sigma-Aldrich) for immunofluorescence; His-tag: mouse monoclonal antibody (Novagen, Darmstadt, Germany), myc-tag: mouse monoclonal antibody (Santa Cruz Biotechnology).

    Techniques: Transfection, Staining

    Transfection with siRNAs against flotillin-2 but not against flotillin-1 reduces autophosphorylation of FAK. HeLa cells were transfected with the indicated siRNAs. The cells were detached, kept in suspension, and then seeded on fibronectin for different times as indicated. Autophosphorylation of FAK was measured by Western blot with a phospho-site-specific antibody (pY397-FAK) in flotillin-1 ( a ) or flotillin-2 ( b ) siRNA-transfected cells. GAPDH and FAK were used as equal loading controls. ( c ) For quantification, the pY397-FAK signal in each sample was first normalized to the total FAK signal. The highest phosphorylation value (control, 25 min FN) was used as the reference and set to 100%, to which all other values were correlated. The bars represent the mean ± SD of at least four independent experiments. For statistical analysis, the significance of each value against the corresponding control value is shown ( n ≥ 4, *** p

    Journal: Cells

    Article Title: Flotillins Regulate Focal Adhesions by Interacting with α-Actinin and by Influencing the Activation of Focal Adhesion Kinase

    doi: 10.3390/cells7040028

    Figure Lengend Snippet: Transfection with siRNAs against flotillin-2 but not against flotillin-1 reduces autophosphorylation of FAK. HeLa cells were transfected with the indicated siRNAs. The cells were detached, kept in suspension, and then seeded on fibronectin for different times as indicated. Autophosphorylation of FAK was measured by Western blot with a phospho-site-specific antibody (pY397-FAK) in flotillin-1 ( a ) or flotillin-2 ( b ) siRNA-transfected cells. GAPDH and FAK were used as equal loading controls. ( c ) For quantification, the pY397-FAK signal in each sample was first normalized to the total FAK signal. The highest phosphorylation value (control, 25 min FN) was used as the reference and set to 100%, to which all other values were correlated. The bars represent the mean ± SD of at least four independent experiments. For statistical analysis, the significance of each value against the corresponding control value is shown ( n ≥ 4, *** p

    Article Snippet: Antibodies Flotillin-1 and flotillin-2: mouse monoclonal (BD Biosciences, Heidelberg, Germany) and rabbit polyclonal (Sigma-Aldrich) antibodies; vinculin: mouse monoclonal antibody (Sigma-Aldrich); FAK and pY397-FAK: mouse monoclonal (BD Biosciences); ERK1/2: rabbit polyclonal (C-14, Santa Cruz Biotechnology, Heidelberg, Germany) antibodies; pERK2: mouse monoclonal antibody (E-4, Santa Cruz); GAPDH: mouse monoclonal antibody (Abcam, Cambridge, UK); α-actinin: rabbit polyclonal H-300 antibody (Santa Cruz) for IP, mouse monoclonal antibody (BD Biosciences) for Western blot and mouse monoclonal antibody (BM-75-1, Sigma-Aldrich) for immunofluorescence; His-tag: mouse monoclonal antibody (Novagen, Darmstadt, Germany), myc-tag: mouse monoclonal antibody (Santa Cruz Biotechnology).

    Techniques: Transfection, Western Blot

    Transfection with siRNAs against flotillin-1 or flotillin-2 reduces ERK2 activation upon integrin stimulation. HeLa cells were transfected with the indicated siRNAs against ( a ) flotillin-1 or ( b ) flotillin-2, or with a control siRNA. The cells were detached, kept in suspension, and then seeded on fibronectin for the indicated time points. ( c ) For quantification, the value for the control siRNA cells (25 min) was used as a reference sample and set to 100%. The phospho- (p)ERK signal in each sample was first normalized to the total ERK signal and then correlated to the reference sample. The bars represent the mean ± SD of at least three independent experiments. For statistical analysis, the significance of each value against the corresponding control value is shown ( n ≥ 3, * p

    Journal: Cells

    Article Title: Flotillins Regulate Focal Adhesions by Interacting with α-Actinin and by Influencing the Activation of Focal Adhesion Kinase

    doi: 10.3390/cells7040028

    Figure Lengend Snippet: Transfection with siRNAs against flotillin-1 or flotillin-2 reduces ERK2 activation upon integrin stimulation. HeLa cells were transfected with the indicated siRNAs against ( a ) flotillin-1 or ( b ) flotillin-2, or with a control siRNA. The cells were detached, kept in suspension, and then seeded on fibronectin for the indicated time points. ( c ) For quantification, the value for the control siRNA cells (25 min) was used as a reference sample and set to 100%. The phospho- (p)ERK signal in each sample was first normalized to the total ERK signal and then correlated to the reference sample. The bars represent the mean ± SD of at least three independent experiments. For statistical analysis, the significance of each value against the corresponding control value is shown ( n ≥ 3, * p

    Article Snippet: Antibodies Flotillin-1 and flotillin-2: mouse monoclonal (BD Biosciences, Heidelberg, Germany) and rabbit polyclonal (Sigma-Aldrich) antibodies; vinculin: mouse monoclonal antibody (Sigma-Aldrich); FAK and pY397-FAK: mouse monoclonal (BD Biosciences); ERK1/2: rabbit polyclonal (C-14, Santa Cruz Biotechnology, Heidelberg, Germany) antibodies; pERK2: mouse monoclonal antibody (E-4, Santa Cruz); GAPDH: mouse monoclonal antibody (Abcam, Cambridge, UK); α-actinin: rabbit polyclonal H-300 antibody (Santa Cruz) for IP, mouse monoclonal antibody (BD Biosciences) for Western blot and mouse monoclonal antibody (BM-75-1, Sigma-Aldrich) for immunofluorescence; His-tag: mouse monoclonal antibody (Novagen, Darmstadt, Germany), myc-tag: mouse monoclonal antibody (Santa Cruz Biotechnology).

    Techniques: Transfection, Activation Assay

    Flotillin-1 and flotillin-2 colocalize with α-actinin in peripheral membrane ruffles. ( a ) MCF10A cells were seeded on coverslips, and endogenous α-actinin and flotillin-1 (upper row) or flotillin-2 (lower row) were visualized with specific antibodies. ( b ) HeLa cells were transfected with the indicated constructs. His–α-actinin was stained with an anti-His-tag antibody. The arrows in the overlay images point to the colocalization of flotillins with α-actinin. Scale bar = 10 µm.

    Journal: Cells

    Article Title: Flotillins Regulate Focal Adhesions by Interacting with α-Actinin and by Influencing the Activation of Focal Adhesion Kinase

    doi: 10.3390/cells7040028

    Figure Lengend Snippet: Flotillin-1 and flotillin-2 colocalize with α-actinin in peripheral membrane ruffles. ( a ) MCF10A cells were seeded on coverslips, and endogenous α-actinin and flotillin-1 (upper row) or flotillin-2 (lower row) were visualized with specific antibodies. ( b ) HeLa cells were transfected with the indicated constructs. His–α-actinin was stained with an anti-His-tag antibody. The arrows in the overlay images point to the colocalization of flotillins with α-actinin. Scale bar = 10 µm.

    Article Snippet: Antibodies Flotillin-1 and flotillin-2: mouse monoclonal (BD Biosciences, Heidelberg, Germany) and rabbit polyclonal (Sigma-Aldrich) antibodies; vinculin: mouse monoclonal antibody (Sigma-Aldrich); FAK and pY397-FAK: mouse monoclonal (BD Biosciences); ERK1/2: rabbit polyclonal (C-14, Santa Cruz Biotechnology, Heidelberg, Germany) antibodies; pERK2: mouse monoclonal antibody (E-4, Santa Cruz); GAPDH: mouse monoclonal antibody (Abcam, Cambridge, UK); α-actinin: rabbit polyclonal H-300 antibody (Santa Cruz) for IP, mouse monoclonal antibody (BD Biosciences) for Western blot and mouse monoclonal antibody (BM-75-1, Sigma-Aldrich) for immunofluorescence; His-tag: mouse monoclonal antibody (Novagen, Darmstadt, Germany), myc-tag: mouse monoclonal antibody (Santa Cruz Biotechnology).

    Techniques: Transfection, Construct, Staining

    Flotillin-2 and α-actinin interaction depends on flotillin-1 expression. Stable control and flotillin-1 knockdown MCF10A cells were grown on 10 cm plates to confluence, lysed, and used for a GST pulldown or coimmunoprecipitation. ( a ) Purified recombinant human α-actinin-1–GST was used to pull down interacting proteins from MCF10A cell lysates, and GST was used as a negative control. The interaction with flotillins was analyzed by Western blot. GAPDH shows equal loading of the lysates and functioned as a negative control for the pulldown. Lowermost blot: Ponceau staining for the fusion proteins (marked with *) used in this assay. ( b ) Cell lysates were precipitated with a polyclonal antibody against α-actinin or mock-precipitated. The immunoprecipitates were analyzed for the presence of α-actinin and coimmunoprecipitated flotillin-1 and flotillin-2 by means of Western blot ( n = 6). Flotillin-2 signals in the immunoprecipitates were quantified and are presented as % of the respective lysate signal normalized for the total protein amount in the lysate.

    Journal: Cells

    Article Title: Flotillins Regulate Focal Adhesions by Interacting with α-Actinin and by Influencing the Activation of Focal Adhesion Kinase

    doi: 10.3390/cells7040028

    Figure Lengend Snippet: Flotillin-2 and α-actinin interaction depends on flotillin-1 expression. Stable control and flotillin-1 knockdown MCF10A cells were grown on 10 cm plates to confluence, lysed, and used for a GST pulldown or coimmunoprecipitation. ( a ) Purified recombinant human α-actinin-1–GST was used to pull down interacting proteins from MCF10A cell lysates, and GST was used as a negative control. The interaction with flotillins was analyzed by Western blot. GAPDH shows equal loading of the lysates and functioned as a negative control for the pulldown. Lowermost blot: Ponceau staining for the fusion proteins (marked with *) used in this assay. ( b ) Cell lysates were precipitated with a polyclonal antibody against α-actinin or mock-precipitated. The immunoprecipitates were analyzed for the presence of α-actinin and coimmunoprecipitated flotillin-1 and flotillin-2 by means of Western blot ( n = 6). Flotillin-2 signals in the immunoprecipitates were quantified and are presented as % of the respective lysate signal normalized for the total protein amount in the lysate.

    Article Snippet: Antibodies Flotillin-1 and flotillin-2: mouse monoclonal (BD Biosciences, Heidelberg, Germany) and rabbit polyclonal (Sigma-Aldrich) antibodies; vinculin: mouse monoclonal antibody (Sigma-Aldrich); FAK and pY397-FAK: mouse monoclonal (BD Biosciences); ERK1/2: rabbit polyclonal (C-14, Santa Cruz Biotechnology, Heidelberg, Germany) antibodies; pERK2: mouse monoclonal antibody (E-4, Santa Cruz); GAPDH: mouse monoclonal antibody (Abcam, Cambridge, UK); α-actinin: rabbit polyclonal H-300 antibody (Santa Cruz) for IP, mouse monoclonal antibody (BD Biosciences) for Western blot and mouse monoclonal antibody (BM-75-1, Sigma-Aldrich) for immunofluorescence; His-tag: mouse monoclonal antibody (Novagen, Darmstadt, Germany), myc-tag: mouse monoclonal antibody (Santa Cruz Biotechnology).

    Techniques: Expressing, Purification, Recombinant, Negative Control, Western Blot, Staining

    Flotillins and α-actinin-1 display Förster Resonance Energy Transfer (FRET) at the cell periphery. ( a ) FRET– Fluorescence Lifetime Imaging (FLIM) analysis was performed with HeLa cells transfected with α-actinin-1–ECFP (FRET donor) alone (lowermost row) or together with flotillin-2–EYFP (uppermost row) or flotillin-1–EYFP (middle row) as acceptors. The reduction of the fluorescence lifetime of the donor was used to calculate FRET efficiencies (see look-up-bar in the upper right corner for color coding). As compared to the τ control, an increased FRET signal is observed, indicated by the appearance of the red color. ( b ) FRET in the donor–acceptor specimen was quantified using a statistical approach. The τ distributions of all donor-only specimen were normalized and averaged. The lower 3-sigma threshold of the donor-only τ distribution was used for determining the percentage of pixels in the donor–acceptor specimen with τ lower than the threshold. The data (mean ± SD) from replicate experiments and images ( n = 5–7) were evaluated for statistical significance using One-Way Anova. ** p ≤ 0.01.

    Journal: Cells

    Article Title: Flotillins Regulate Focal Adhesions by Interacting with α-Actinin and by Influencing the Activation of Focal Adhesion Kinase

    doi: 10.3390/cells7040028

    Figure Lengend Snippet: Flotillins and α-actinin-1 display Förster Resonance Energy Transfer (FRET) at the cell periphery. ( a ) FRET– Fluorescence Lifetime Imaging (FLIM) analysis was performed with HeLa cells transfected with α-actinin-1–ECFP (FRET donor) alone (lowermost row) or together with flotillin-2–EYFP (uppermost row) or flotillin-1–EYFP (middle row) as acceptors. The reduction of the fluorescence lifetime of the donor was used to calculate FRET efficiencies (see look-up-bar in the upper right corner for color coding). As compared to the τ control, an increased FRET signal is observed, indicated by the appearance of the red color. ( b ) FRET in the donor–acceptor specimen was quantified using a statistical approach. The τ distributions of all donor-only specimen were normalized and averaged. The lower 3-sigma threshold of the donor-only τ distribution was used for determining the percentage of pixels in the donor–acceptor specimen with τ lower than the threshold. The data (mean ± SD) from replicate experiments and images ( n = 5–7) were evaluated for statistical significance using One-Way Anova. ** p ≤ 0.01.

    Article Snippet: Antibodies Flotillin-1 and flotillin-2: mouse monoclonal (BD Biosciences, Heidelberg, Germany) and rabbit polyclonal (Sigma-Aldrich) antibodies; vinculin: mouse monoclonal antibody (Sigma-Aldrich); FAK and pY397-FAK: mouse monoclonal (BD Biosciences); ERK1/2: rabbit polyclonal (C-14, Santa Cruz Biotechnology, Heidelberg, Germany) antibodies; pERK2: mouse monoclonal antibody (E-4, Santa Cruz); GAPDH: mouse monoclonal antibody (Abcam, Cambridge, UK); α-actinin: rabbit polyclonal H-300 antibody (Santa Cruz) for IP, mouse monoclonal antibody (BD Biosciences) for Western blot and mouse monoclonal antibody (BM-75-1, Sigma-Aldrich) for immunofluorescence; His-tag: mouse monoclonal antibody (Novagen, Darmstadt, Germany), myc-tag: mouse monoclonal antibody (Santa Cruz Biotechnology).

    Techniques: Förster Resonance Energy Transfer, Fluorescence, Imaging, Transfection

    Flotillin knockdown cells display a reduced migration rate in a wound healing assay, and depletion of flotillins results in impaired haptotactic migration, slower cell spreading and reduced number of FAs. ( a ) HeLa cells transfected with the indicated siRNAs were allowed to grow until confluent. A defined scratch was then produced (0 h, upper panels), and the closure of the wounded area was monitored over 24 h (lower panels). The photographs show a representative section from n ≥ 3 experiments. The graphs represent plot profiles with integrated pixel density across the wound area. ( b ) HeLa cells were transfected with the indicated siRNAs. The lower side of a Transwell membrane was coated with fibronectin, and the cells were seeded in the upper part. After 6 h, the amount of migrated cells on the lower membrane part was measured. The control siRNA sample was used as the reference value and set to 100%. At least five independent experiments with duplicates per sample were performed ( n ≥ 5, ** p

    Journal: Cells

    Article Title: Flotillins Regulate Focal Adhesions by Interacting with α-Actinin and by Influencing the Activation of Focal Adhesion Kinase

    doi: 10.3390/cells7040028

    Figure Lengend Snippet: Flotillin knockdown cells display a reduced migration rate in a wound healing assay, and depletion of flotillins results in impaired haptotactic migration, slower cell spreading and reduced number of FAs. ( a ) HeLa cells transfected with the indicated siRNAs were allowed to grow until confluent. A defined scratch was then produced (0 h, upper panels), and the closure of the wounded area was monitored over 24 h (lower panels). The photographs show a representative section from n ≥ 3 experiments. The graphs represent plot profiles with integrated pixel density across the wound area. ( b ) HeLa cells were transfected with the indicated siRNAs. The lower side of a Transwell membrane was coated with fibronectin, and the cells were seeded in the upper part. After 6 h, the amount of migrated cells on the lower membrane part was measured. The control siRNA sample was used as the reference value and set to 100%. At least five independent experiments with duplicates per sample were performed ( n ≥ 5, ** p

    Article Snippet: Antibodies Flotillin-1 and flotillin-2: mouse monoclonal (BD Biosciences, Heidelberg, Germany) and rabbit polyclonal (Sigma-Aldrich) antibodies; vinculin: mouse monoclonal antibody (Sigma-Aldrich); FAK and pY397-FAK: mouse monoclonal (BD Biosciences); ERK1/2: rabbit polyclonal (C-14, Santa Cruz Biotechnology, Heidelberg, Germany) antibodies; pERK2: mouse monoclonal antibody (E-4, Santa Cruz); GAPDH: mouse monoclonal antibody (Abcam, Cambridge, UK); α-actinin: rabbit polyclonal H-300 antibody (Santa Cruz) for IP, mouse monoclonal antibody (BD Biosciences) for Western blot and mouse monoclonal antibody (BM-75-1, Sigma-Aldrich) for immunofluorescence; His-tag: mouse monoclonal antibody (Novagen, Darmstadt, Germany), myc-tag: mouse monoclonal antibody (Santa Cruz Biotechnology).

    Techniques: Migration, Wound Healing Assay, Transfection, Produced

    Knockdown of flotillins reduces anchorage-independent growth in soft agar, whereas overexpression results in larger colonies. ( a ) HeLa cells transfected with the indicated siRNAs were seeded into soft agar and allowed to grow for 14 days, after which the colonies were counted ( b ), and their average size determined ( c ). The bars show mean ± SD ( n ≥ 4, * p

    Journal: Cells

    Article Title: Flotillins Regulate Focal Adhesions by Interacting with α-Actinin and by Influencing the Activation of Focal Adhesion Kinase

    doi: 10.3390/cells7040028

    Figure Lengend Snippet: Knockdown of flotillins reduces anchorage-independent growth in soft agar, whereas overexpression results in larger colonies. ( a ) HeLa cells transfected with the indicated siRNAs were seeded into soft agar and allowed to grow for 14 days, after which the colonies were counted ( b ), and their average size determined ( c ). The bars show mean ± SD ( n ≥ 4, * p

    Article Snippet: Antibodies Flotillin-1 and flotillin-2: mouse monoclonal (BD Biosciences, Heidelberg, Germany) and rabbit polyclonal (Sigma-Aldrich) antibodies; vinculin: mouse monoclonal antibody (Sigma-Aldrich); FAK and pY397-FAK: mouse monoclonal (BD Biosciences); ERK1/2: rabbit polyclonal (C-14, Santa Cruz Biotechnology, Heidelberg, Germany) antibodies; pERK2: mouse monoclonal antibody (E-4, Santa Cruz); GAPDH: mouse monoclonal antibody (Abcam, Cambridge, UK); α-actinin: rabbit polyclonal H-300 antibody (Santa Cruz) for IP, mouse monoclonal antibody (BD Biosciences) for Western blot and mouse monoclonal antibody (BM-75-1, Sigma-Aldrich) for immunofluorescence; His-tag: mouse monoclonal antibody (Novagen, Darmstadt, Germany), myc-tag: mouse monoclonal antibody (Santa Cruz Biotechnology).

    Techniques: Over Expression, Transfection

    Role of Toll-like receptor 4 (TLR4) in the neuroprotection by NTP. ( A ) Effect of TAK-242 on NTP-induced formation of URFs. PCtrk cells (5 × 10 6 cells) were treated in serum-free DMEM containing saline control (open symbols, or CRL), NTP (20 mNU/mL, closed symbols), or NTP and TAK-242 (10 nM, gray symbols, or TAK) for 3 h, lysed in 0.5% Triton X-100-containing TNE buffer and centrifuged in sucrose density gradients as described in Materials and Methods. Cholesterol levels in each fraction were quantified by Amplex Red Cholesterol Assay Kit to confirm peaks of the lipid rafts and URFs. Representative data are shown. Moreover, we also analyzed a rafts-marker protein, flotillin-1 distribution in sucrose density gradient fractionation of the cells treated with each treatment. NTP-treatment caused new emergence of flotillin-1 in URFs and it was abolished by TAK-242 treatment. ( B ) Cholesterol contents in the peak fraction of typical lipid rafts (Panel a , fraction no.3) and of URFs (Panel b , fraction no. 7) are summarized as mean and SD of three independent experiments. ns, not significant; ** p

    Journal: International Journal of Molecular Sciences

    Article Title: Neuroprotection by Neurotropin through Crosstalk of Neurotrophic and Innate Immune Receptors in PC12 Cells

    doi: 10.3390/ijms21186456

    Figure Lengend Snippet: Role of Toll-like receptor 4 (TLR4) in the neuroprotection by NTP. ( A ) Effect of TAK-242 on NTP-induced formation of URFs. PCtrk cells (5 × 10 6 cells) were treated in serum-free DMEM containing saline control (open symbols, or CRL), NTP (20 mNU/mL, closed symbols), or NTP and TAK-242 (10 nM, gray symbols, or TAK) for 3 h, lysed in 0.5% Triton X-100-containing TNE buffer and centrifuged in sucrose density gradients as described in Materials and Methods. Cholesterol levels in each fraction were quantified by Amplex Red Cholesterol Assay Kit to confirm peaks of the lipid rafts and URFs. Representative data are shown. Moreover, we also analyzed a rafts-marker protein, flotillin-1 distribution in sucrose density gradient fractionation of the cells treated with each treatment. NTP-treatment caused new emergence of flotillin-1 in URFs and it was abolished by TAK-242 treatment. ( B ) Cholesterol contents in the peak fraction of typical lipid rafts (Panel a , fraction no.3) and of URFs (Panel b , fraction no. 7) are summarized as mean and SD of three independent experiments. ns, not significant; ** p

    Article Snippet: Antibodies used for Western blotting were as follows: anti-Trk antibody (C-14), anti-Fyn antibody (sc-434), anti-phosphotyrosine antibody (clone 4G10; Millipore; RRID: AB_310802), anti-NF-M antibody (NA1216; Affiniti Research, Devon, UK; RRID: AB_10541956), anti-TfR(H68.4; Invitrogen; RRID: AB_86624), anti-TLR4 (sc-293072; Santa Cruz Biotechnology, Santa Cruz, CA, USA; PRID: AB_10611320; α-TLR4), anti-flotillin-1 antibody (610810; BD Transduction Laboratories, San Jose, CA, USA; RRID: AB_398189) and anti-β-actin antibody (clone 4967; Cell Signaling Technology, Danvers, MA, USA; RRID: AB_330288) for primary antibodies and anti-rabbit IgG (AP132P; Chemicon International, Temecula, CA, USA; RRID: AB_90264) and anti-mouse IgG (NA931; Amersham Bioscience, Buckinghamshire, UK; RRID: AB_772210) for secondary antibodies conjugated with HRP.

    Techniques: Amplex Red Cholesterol Assay, Marker, Fractionation

    Subcellular distribution of lipid rafts in PCtrk cells treated with NTP. PCtrk cells (5 × 10 6 cells) were treated with 20 mNU/mL NTP (closed symbols, or NTP) or saline (open symbols, or CRL) for 3 h, lysed in 0.5% Triton X-100-containing TNE buffer and centrifuged in sucrose density gradients to isolate lipid rafts, as described in Materials and Methods. Representative data are shown. ( A ) Cholesterol concentrations determined by Amplex Red Cholesterol Assay Kit. ( B ) GM1 ± fucosyl-GM1 concentrations determined by dot blot analysis using cholera toxin subunit B subunit- conjugated with horseradish peroxidase. ( C ) Distribution of transferrin receptor (TfR, 95 KDa), Trk (140 KDa), Fyn (59 KDa), Toll-like receptor TLR4 (95 KDa), and flotillin-1 (47 KDa) determined by Western blotting. Detergent-insoluble typical lipid rafts were fractionated in this condition into fractions 2–5. Note that NTP treatment formed novel membrane fractions (no. 6–8) with high-buoyant density (unidentified raft-like fractions, URFs). (D) Statistical analysis of the Trk protein distribution in lipid rafts and URFs fraction. We measured the densities of each band corresponding to Trk in fractions by ImageJ software (ver. 1.51; NIH, USA; RRID: SCR_003070). Then, we calculated the ratio of densities of each fraction corresponding to rafts (fraction No. 2–5; columns 2 and 5, n = 4) and URFs (fraction No. 6–8; columns 3 and 6, n = 4) against that of fraction No. 1 (columns 1 and 4; this fraction did not contain Trk protein but exhibit background densities, n = 4) and further calculated the mean ratio ± SE of rafts fractions and of URFs. These data were subjected to the statistical analysis (Wilcoxon ranked sum test) * p

    Journal: International Journal of Molecular Sciences

    Article Title: Neuroprotection by Neurotropin through Crosstalk of Neurotrophic and Innate Immune Receptors in PC12 Cells

    doi: 10.3390/ijms21186456

    Figure Lengend Snippet: Subcellular distribution of lipid rafts in PCtrk cells treated with NTP. PCtrk cells (5 × 10 6 cells) were treated with 20 mNU/mL NTP (closed symbols, or NTP) or saline (open symbols, or CRL) for 3 h, lysed in 0.5% Triton X-100-containing TNE buffer and centrifuged in sucrose density gradients to isolate lipid rafts, as described in Materials and Methods. Representative data are shown. ( A ) Cholesterol concentrations determined by Amplex Red Cholesterol Assay Kit. ( B ) GM1 ± fucosyl-GM1 concentrations determined by dot blot analysis using cholera toxin subunit B subunit- conjugated with horseradish peroxidase. ( C ) Distribution of transferrin receptor (TfR, 95 KDa), Trk (140 KDa), Fyn (59 KDa), Toll-like receptor TLR4 (95 KDa), and flotillin-1 (47 KDa) determined by Western blotting. Detergent-insoluble typical lipid rafts were fractionated in this condition into fractions 2–5. Note that NTP treatment formed novel membrane fractions (no. 6–8) with high-buoyant density (unidentified raft-like fractions, URFs). (D) Statistical analysis of the Trk protein distribution in lipid rafts and URFs fraction. We measured the densities of each band corresponding to Trk in fractions by ImageJ software (ver. 1.51; NIH, USA; RRID: SCR_003070). Then, we calculated the ratio of densities of each fraction corresponding to rafts (fraction No. 2–5; columns 2 and 5, n = 4) and URFs (fraction No. 6–8; columns 3 and 6, n = 4) against that of fraction No. 1 (columns 1 and 4; this fraction did not contain Trk protein but exhibit background densities, n = 4) and further calculated the mean ratio ± SE of rafts fractions and of URFs. These data were subjected to the statistical analysis (Wilcoxon ranked sum test) * p

    Article Snippet: Antibodies used for Western blotting were as follows: anti-Trk antibody (C-14), anti-Fyn antibody (sc-434), anti-phosphotyrosine antibody (clone 4G10; Millipore; RRID: AB_310802), anti-NF-M antibody (NA1216; Affiniti Research, Devon, UK; RRID: AB_10541956), anti-TfR(H68.4; Invitrogen; RRID: AB_86624), anti-TLR4 (sc-293072; Santa Cruz Biotechnology, Santa Cruz, CA, USA; PRID: AB_10611320; α-TLR4), anti-flotillin-1 antibody (610810; BD Transduction Laboratories, San Jose, CA, USA; RRID: AB_398189) and anti-β-actin antibody (clone 4967; Cell Signaling Technology, Danvers, MA, USA; RRID: AB_330288) for primary antibodies and anti-rabbit IgG (AP132P; Chemicon International, Temecula, CA, USA; RRID: AB_90264) and anti-mouse IgG (NA931; Amersham Bioscience, Buckinghamshire, UK; RRID: AB_772210) for secondary antibodies conjugated with HRP.

    Techniques: Amplex Red Cholesterol Assay, Dot Blot, Western Blot, Software

    Cholesterol depletion does not inhibit heregulin-dependent translocation of ErbB-4 to a Triton-insoluble fraction. T47D cells were pretreated treated with either methyl-β-cyclodextrin (5mM) or α-cyclodextrin (5mM) for 30 min. followed by heregulin (20 ng/ml) for an additional 15 min. and separated into Triton X-100-soluble and insoluble fractions. The Triton-insoluble fractions were immunoprecipitated with anti-ErbB-4 and blotted for ErbB-4 (upper panel). Lysates were blotted for flotillin-1 expression (lower panel).

    Journal: Biochemical and biophysical research communications

    Article Title: ErbB-4 and TNF-? Converting Enzyme Localization to Membrane Microdomains

    doi: 10.1016/j.bbrc.2006.09.095

    Figure Lengend Snippet: Cholesterol depletion does not inhibit heregulin-dependent translocation of ErbB-4 to a Triton-insoluble fraction. T47D cells were pretreated treated with either methyl-β-cyclodextrin (5mM) or α-cyclodextrin (5mM) for 30 min. followed by heregulin (20 ng/ml) for an additional 15 min. and separated into Triton X-100-soluble and insoluble fractions. The Triton-insoluble fractions were immunoprecipitated with anti-ErbB-4 and blotted for ErbB-4 (upper panel). Lysates were blotted for flotillin-1 expression (lower panel).

    Article Snippet: ErbB-4 antibody (C-18) was purchased from Santa Cruz Biotechnologies, TACE antibody (807–823) from Calbiochem, and flotillin antibody from BD Transduction Laboratories.

    Techniques: Translocation Assay, Immunoprecipitation, Expressing

    Heregulin-induced ErbB-4 translocation to a Triton X-100-insoluble fraction. T47D (Panel A) or COS-7 (Panel B) ]. Equal volumes of soluble and insoluble fractions were either immunoprecipitated with anti-ErbB-4 (upper panels) or directly subjected to SDS-PAGE and blotted with antibodies directed against ErbB-4, TACE or flotillin-1. ErbB-4 bands in the Triton-insoluble fraction were subjected to densitometric analysis, normalized for flotillin-1 levels, and quantitated relative to untreated samples. Arrows indicate immature (upper arrow, 120 kDa) and mature (lower arrow, 100 kDa) forms of TACE (center panels).

    Journal: Biochemical and biophysical research communications

    Article Title: ErbB-4 and TNF-? Converting Enzyme Localization to Membrane Microdomains

    doi: 10.1016/j.bbrc.2006.09.095

    Figure Lengend Snippet: Heregulin-induced ErbB-4 translocation to a Triton X-100-insoluble fraction. T47D (Panel A) or COS-7 (Panel B) ]. Equal volumes of soluble and insoluble fractions were either immunoprecipitated with anti-ErbB-4 (upper panels) or directly subjected to SDS-PAGE and blotted with antibodies directed against ErbB-4, TACE or flotillin-1. ErbB-4 bands in the Triton-insoluble fraction were subjected to densitometric analysis, normalized for flotillin-1 levels, and quantitated relative to untreated samples. Arrows indicate immature (upper arrow, 120 kDa) and mature (lower arrow, 100 kDa) forms of TACE (center panels).

    Article Snippet: ErbB-4 antibody (C-18) was purchased from Santa Cruz Biotechnologies, TACE antibody (807–823) from Calbiochem, and flotillin antibody from BD Transduction Laboratories.

    Techniques: Translocation Assay, Immunoprecipitation, SDS Page

    Detergent-free lipid raft isolation. (Panel A) T47D cells were starved overnight, treated with heregulin (20ng/ml) for 15 min. and lipid rafts isolated by the detergent-free method of Smart et al. ]. Equal aliquots of lipid raft fractions were blotted with anti-ErbB-4 (upper panel), anti-TACE (center panel) and anti-flotillin-1 (lower panel). (Panel B) Lipid rafts from COS-7 cells expressing JM-a or JM-b ErbB-4 were isolated and analyzed for ErbB-4, TACE, and flotillin-1 localization as in Panel A. Results reflecting two experiments analyzed in duplicate were normalized for flotillin-1 levels and quantitated on the right.

    Journal: Biochemical and biophysical research communications

    Article Title: ErbB-4 and TNF-? Converting Enzyme Localization to Membrane Microdomains

    doi: 10.1016/j.bbrc.2006.09.095

    Figure Lengend Snippet: Detergent-free lipid raft isolation. (Panel A) T47D cells were starved overnight, treated with heregulin (20ng/ml) for 15 min. and lipid rafts isolated by the detergent-free method of Smart et al. ]. Equal aliquots of lipid raft fractions were blotted with anti-ErbB-4 (upper panel), anti-TACE (center panel) and anti-flotillin-1 (lower panel). (Panel B) Lipid rafts from COS-7 cells expressing JM-a or JM-b ErbB-4 were isolated and analyzed for ErbB-4, TACE, and flotillin-1 localization as in Panel A. Results reflecting two experiments analyzed in duplicate were normalized for flotillin-1 levels and quantitated on the right.

    Article Snippet: ErbB-4 antibody (C-18) was purchased from Santa Cruz Biotechnologies, TACE antibody (807–823) from Calbiochem, and flotillin antibody from BD Transduction Laboratories.

    Techniques: Isolation, Expressing

    The effect of lovastatin and MβCD treatment on APP cleavage by GPI-BACE. Confluent SH-SY5Y cells stably expressing WT-BACE or GPI-BACE were treated with lovastatin and methyl-β-cyclodextrin as described in Experimental Procedures , or incubated in OptiMEM for equal lengths of time. The medium was collected, and lipid rafts were prepared as described. ( A ) Sucrose gradients were harvested in 0.5-ml fractions (fraction 1, top of gradient; fraction 9, bottom of gradient), and each fraction was analyzed for BACE and flotillin by SDS/PAGE and immunoblotting with the monoclonal 9B21 BACE antibody or a monoclonal flotillin antibody. ( B ) Media collected from WTBACE- or GPI-BACE-expressing cells incubated in the absence or presence of lovastatin was concentrated, and equal amounts of protein were analyzed by SDS/PAGE and immunoblotting with the G26 antibody to sAPPβ. Densitometric analysis was carried out, and the mean results ± SEM ( n = 3) are shown graphically (black bars, untreated cells; gray bars, treated cells).

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Exclusively targeting ?-secretase to lipid rafts by GPI-anchor addition up-regulates ?-site processing of the amyloid precursor protein

    doi: 10.1073/pnas.1635130100

    Figure Lengend Snippet: The effect of lovastatin and MβCD treatment on APP cleavage by GPI-BACE. Confluent SH-SY5Y cells stably expressing WT-BACE or GPI-BACE were treated with lovastatin and methyl-β-cyclodextrin as described in Experimental Procedures , or incubated in OptiMEM for equal lengths of time. The medium was collected, and lipid rafts were prepared as described. ( A ) Sucrose gradients were harvested in 0.5-ml fractions (fraction 1, top of gradient; fraction 9, bottom of gradient), and each fraction was analyzed for BACE and flotillin by SDS/PAGE and immunoblotting with the monoclonal 9B21 BACE antibody or a monoclonal flotillin antibody. ( B ) Media collected from WTBACE- or GPI-BACE-expressing cells incubated in the absence or presence of lovastatin was concentrated, and equal amounts of protein were analyzed by SDS/PAGE and immunoblotting with the G26 antibody to sAPPβ. Densitometric analysis was carried out, and the mean results ± SEM ( n = 3) are shown graphically (black bars, untreated cells; gray bars, treated cells).

    Article Snippet: The antibodies against flotillin (BD Biosciences) and β-actin (Sigma) were used at 1:250 and 1:5,000, respectively.

    Techniques: Stable Transfection, Expressing, Incubation, SDS Page

    Isolation of lipid rafts from SH-SY5Y cells stably expressing WT-BACE or GPI-BACE. Lipid rafts were prepared as described in Experimental Procedures from cells stably expressing WT-BACE ( A ) or GPI-BACE ( B ). Sucrose gradients were harvested in 0.5-ml fractions (fraction 1, top of gradient; fraction 9, bottom of gradient), and each fraction was analyzed by SDS/PAGE and immunoblotted with the monoclonal 9B21 BACE antibody or a monoclonal flotillin antibody.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Exclusively targeting ?-secretase to lipid rafts by GPI-anchor addition up-regulates ?-site processing of the amyloid precursor protein

    doi: 10.1073/pnas.1635130100

    Figure Lengend Snippet: Isolation of lipid rafts from SH-SY5Y cells stably expressing WT-BACE or GPI-BACE. Lipid rafts were prepared as described in Experimental Procedures from cells stably expressing WT-BACE ( A ) or GPI-BACE ( B ). Sucrose gradients were harvested in 0.5-ml fractions (fraction 1, top of gradient; fraction 9, bottom of gradient), and each fraction was analyzed by SDS/PAGE and immunoblotted with the monoclonal 9B21 BACE antibody or a monoclonal flotillin antibody.

    Article Snippet: The antibodies against flotillin (BD Biosciences) and β-actin (Sigma) were used at 1:250 and 1:5,000, respectively.

    Techniques: Isolation, Stable Transfection, Expressing, SDS Page

    Verification of EV isolation. Laemmli and TRIzol©-extracted samples were interrogated by Western blot for the expression of the canonical EV markers HSP-70, CD63, Flotillin-1, CD81 and CD9. MCF-7 (7), MCF-10A (10A) and MDA-MB-231 (231) cell EVs were examined. HSP-70 (top), CD63 (middle) and Flotillin-1 (bottom) were detected in the isolated EVs.

    Journal: Journal of Extracellular Vesicles

    Article Title: Proteome profiling of extracellular vesicles captured with the affinity peptide Vn96: comparison of Laemmli and TRIzol© protein-extraction methods

    doi: 10.1080/20013078.2018.1438727

    Figure Lengend Snippet: Verification of EV isolation. Laemmli and TRIzol©-extracted samples were interrogated by Western blot for the expression of the canonical EV markers HSP-70, CD63, Flotillin-1, CD81 and CD9. MCF-7 (7), MCF-10A (10A) and MDA-MB-231 (231) cell EVs were examined. HSP-70 (top), CD63 (middle) and Flotillin-1 (bottom) were detected in the isolated EVs.

    Article Snippet: Anti-human antibodies (from Santa Cruz, Santa Cruz, CA, unless otherwise specified) were prepared in TBST with 5% BSA and incubated overnight at 4°C as follows: 1:200 HSC-70 (SC-7298), 1:1000 Flotillin-1 (Cell Signaling Technology, Danvers, MA; 18,634), 1:200 CD63 (SC-5275), 1:200 CD9 (SC-59,140), 1:1000 CD81 (SC-7637).

    Techniques: Isolation, Western Blot, Expressing, Multiple Displacement Amplification

    Stat3-mediated regulation of the lysosomal compartment. A , validation by Western blot analysis of the lysosomal localization of flotillin 1 and flotillin 2 using iron nanoparticles to isolate EpH4 lysosomes (Mag+) after 72 h of OSM stimulation. White asterisk , indicates the expected molecular weight of flotillin 2. Representative blots of n = 3 (flotillin 1) and n = 2 (flotillin 2) independent experiments are shown. B , microarray analysis of 12 different timepoints during the mammary gland pregnancy cycle showing the involution-related expression profiles of flotillin 1 and flotillin 2. V , virgin; d G , days gestation; d L , days lactation; h I , hours involution. C , fluorescence microscopy of LAMP1 and LAMP2 immunostaining in EpH4 cells treated with OSM for 72 h. Scale bars : 10 μm. D , Western blot analysis of OSM-induced changes to the lysosomal proteins LAMP1, LAMP2, CD63, and cathepsin B in whole-cell lysates from normal (WT) and two independent Stat3 KO EpH4 cell lines after 72 h of stimulation. Proteins were separated by SDS-PAGE under reducing or non-reducing conditions as indicated. pro-Ctsb , pro form of cathepsin B; sc Ctsb , single-chain cathepsin B; Scram , scrambled sgRNA control EpH4 cells. E , microarray analysis of 12 different timepoints during the mammary gland pregnancy cycle showing the involution-related expression profiles of CD63 and LAMP2.

    Journal: The Journal of Biological Chemistry

    Article Title: Stat3-mediated alterations in lysosomal membrane protein composition

    doi: 10.1074/jbc.RA118.001777

    Figure Lengend Snippet: Stat3-mediated regulation of the lysosomal compartment. A , validation by Western blot analysis of the lysosomal localization of flotillin 1 and flotillin 2 using iron nanoparticles to isolate EpH4 lysosomes (Mag+) after 72 h of OSM stimulation. White asterisk , indicates the expected molecular weight of flotillin 2. Representative blots of n = 3 (flotillin 1) and n = 2 (flotillin 2) independent experiments are shown. B , microarray analysis of 12 different timepoints during the mammary gland pregnancy cycle showing the involution-related expression profiles of flotillin 1 and flotillin 2. V , virgin; d G , days gestation; d L , days lactation; h I , hours involution. C , fluorescence microscopy of LAMP1 and LAMP2 immunostaining in EpH4 cells treated with OSM for 72 h. Scale bars : 10 μm. D , Western blot analysis of OSM-induced changes to the lysosomal proteins LAMP1, LAMP2, CD63, and cathepsin B in whole-cell lysates from normal (WT) and two independent Stat3 KO EpH4 cell lines after 72 h of stimulation. Proteins were separated by SDS-PAGE under reducing or non-reducing conditions as indicated. pro-Ctsb , pro form of cathepsin B; sc Ctsb , single-chain cathepsin B; Scram , scrambled sgRNA control EpH4 cells. E , microarray analysis of 12 different timepoints during the mammary gland pregnancy cycle showing the involution-related expression profiles of CD63 and LAMP2.

    Article Snippet: The following primary antibodies were used: rat anti-LAMP2 (ab13524, Abcam, 1:1,000), goat anti-cathepsin L (AF1515, R & D Systems, 1:1,000), goat anti-cathepsin B (AF965, R & D Systems, 1:1,000), mouse anti-COX IV (ab33985, Abcam, 1:1,000), rabbit anti-phosphatidylinositol 3-kinase, p85 (06-496, Millipore, 1:1000), rabbit anti-Stat3 (sc-482, Santa Cruz Biotechnology, 1:1000), rabbit anti-phospho-Stat3 (9131, Cell Signaling, 1:1000), mouse anti-ERp57 (sc-23886, Santa Cruz Biotechnology, 1:400), rabbit anti-GAPDH (5174, Cell Signaling, 1:1000), goat anti-histone H3 (sc-8654, Santa Cruz Biotechnology, 1:1000), rabbit anti-Rab5 (2143, Cell Signaling, 1:1000), rat anti-LAMP1 (ID4B, Developmental Studies Hybridoma Bank, 1:200), rat anti-CD63 (NVG-2, 143901, Biolegend, 1:1000), rabbit anti-flotillin 1 (18634, Cell Signaling Technology, 1:800), and rabbit anti-flotillin 2 (HPA0013961, Atlas Antibodies, 1:200).

    Techniques: Western Blot, Molecular Weight, Microarray, Expressing, Fluorescence, Microscopy, Immunostaining, SDS Page

    Flotillin depletion increases diffusion mobility rates of CFP-HA-DAT

    Journal: Traffic (Copenhagen, Denmark)

    Article Title: Flotillins regulate membrane mobility of the dopamine transporter but are not required for its protein kinase C dependent endocytosis

    doi: 10.1111/tra.12059

    Figure Lengend Snippet: Flotillin depletion increases diffusion mobility rates of CFP-HA-DAT

    Article Snippet: Flotillin-1 defines a clathrin-independent endocytic pathway in mammalian cells.

    Techniques: Diffusion-based Assay

    Co-immunoprecipitation of DAT with flotillin-1 and Eps15

    Journal: Traffic (Copenhagen, Denmark)

    Article Title: Flotillins regulate membrane mobility of the dopamine transporter but are not required for its protein kinase C dependent endocytosis

    doi: 10.1111/tra.12059

    Figure Lengend Snippet: Co-immunoprecipitation of DAT with flotillin-1 and Eps15

    Article Snippet: Flotillin-1 defines a clathrin-independent endocytic pathway in mammalian cells.

    Techniques: Immunoprecipitation

    PMA-induced down-regulation of DAT in HEK293 cells is blocked by clathrin but not affected by flotillin knockdowns

    Journal: Traffic (Copenhagen, Denmark)

    Article Title: Flotillins regulate membrane mobility of the dopamine transporter but are not required for its protein kinase C dependent endocytosis

    doi: 10.1111/tra.12059

    Figure Lengend Snippet: PMA-induced down-regulation of DAT in HEK293 cells is blocked by clathrin but not affected by flotillin knockdowns

    Article Snippet: Flotillin-1 defines a clathrin-independent endocytic pathway in mammalian cells.

    Techniques:

    Comparison of localization of flotillin-1 and DAT in dopaminergic neurons

    Journal: Traffic (Copenhagen, Denmark)

    Article Title: Flotillins regulate membrane mobility of the dopamine transporter but are not required for its protein kinase C dependent endocytosis

    doi: 10.1111/tra.12059

    Figure Lengend Snippet: Comparison of localization of flotillin-1 and DAT in dopaminergic neurons

    Article Snippet: Flotillin-1 defines a clathrin-independent endocytic pathway in mammalian cells.

    Techniques:

    Depletion of both flotillin-1 and flotillin-2 does not inhibit PMA-dependent endocytosis of DAT in HEK293 cells

    Journal: Traffic (Copenhagen, Denmark)

    Article Title: Flotillins regulate membrane mobility of the dopamine transporter but are not required for its protein kinase C dependent endocytosis

    doi: 10.1111/tra.12059

    Figure Lengend Snippet: Depletion of both flotillin-1 and flotillin-2 does not inhibit PMA-dependent endocytosis of DAT in HEK293 cells

    Article Snippet: Flotillin-1 defines a clathrin-independent endocytic pathway in mammalian cells.

    Techniques:

    Depletion of flotillin-1 and flotillin-2 does not inhibit PMA-dependent endocytosis of DAT in HEK/DAT cells

    Journal: Traffic (Copenhagen, Denmark)

    Article Title: Flotillins regulate membrane mobility of the dopamine transporter but are not required for its protein kinase C dependent endocytosis

    doi: 10.1111/tra.12059

    Figure Lengend Snippet: Depletion of flotillin-1 and flotillin-2 does not inhibit PMA-dependent endocytosis of DAT in HEK/DAT cells

    Article Snippet: Flotillin-1 defines a clathrin-independent endocytic pathway in mammalian cells.

    Techniques:

    Flotillin-1 siRNA does not inhibit PMA-dependent endocytosis of DAT in HeLa cells

    Journal: Traffic (Copenhagen, Denmark)

    Article Title: Flotillins regulate membrane mobility of the dopamine transporter but are not required for its protein kinase C dependent endocytosis

    doi: 10.1111/tra.12059

    Figure Lengend Snippet: Flotillin-1 siRNA does not inhibit PMA-dependent endocytosis of DAT in HeLa cells

    Article Snippet: Flotillin-1 defines a clathrin-independent endocytic pathway in mammalian cells.

    Techniques:

    Comparison of localization of flotillin-1 and DAT in HEK293 cells

    Journal: Traffic (Copenhagen, Denmark)

    Article Title: Flotillins regulate membrane mobility of the dopamine transporter but are not required for its protein kinase C dependent endocytosis

    doi: 10.1111/tra.12059

    Figure Lengend Snippet: Comparison of localization of flotillin-1 and DAT in HEK293 cells

    Article Snippet: Flotillin-1 defines a clathrin-independent endocytic pathway in mammalian cells.

    Techniques:

    Flotillin-1 siRNA does not inhibit PMA-dependent endocytosis of DAT in HEK293 cells

    Journal: Traffic (Copenhagen, Denmark)

    Article Title: Flotillins regulate membrane mobility of the dopamine transporter but are not required for its protein kinase C dependent endocytosis

    doi: 10.1111/tra.12059

    Figure Lengend Snippet: Flotillin-1 siRNA does not inhibit PMA-dependent endocytosis of DAT in HEK293 cells

    Article Snippet: Flotillin-1 defines a clathrin-independent endocytic pathway in mammalian cells.

    Techniques:

    GPCR activation triggers MVB–PM fusion in HeLa cells via SNAP23-Ser110 phosphorylation. (a) Fusion activity of histamine-stimulated cells nontreated or treated with the Gα q inhibitor UBO-QIC (1 µM). n ≥ 24 cells per condition. (b) Basal fusion activity in cells treated with PKC inhibitors GÖ6976 (1 µM) or GÖ6983 (1 µM). n ≥ 11 cells per condition. (c) Fusion activity of histamine-stimulated cells nontreated or preincubated with GÖ6983 (1 µM). n ≥ 11 cells per condition. (d) Schematic representation of SNAP23 with SNARE motifs, a membrane-anchoring domain (M), and all phosphosites with the posphosite targeted by histamine stimulation (Ser110) in bold. (e) Fusion activity of histamine-stimulated cells transfected with WT SNAP23, phosphomutant SNAP23-S110A, or phosphomimic SNAP23-S110D. n ≥ 16 cells per condition. (f) Left: fusion activity of CD63-, CD81-, and CD9-pHluorin HeLa cells cotransfected with SNAP23 WT or SNAP23-S110A. n ≥ 16 cells per condition. Western blot on exosomes isolated from SNAP23-WT and SNAP23-S110A HeLa cells labeled for CD63, CD9, CD81, flotillin-1, and syntenin-1. (g) Schematic representation of the histamine-stimulated pathway leading to exosome release as identified by phosphoproteomics and specific inhibitors. Blue-rimmed proteins represent the putative pathway implicated by both experiments. The IP3–Ca 2+ pathway is represented in gray as a direct link with MVB–PM fusion is missing. **, P

    Journal: The Journal of Cell Biology

    Article Title: Quantifying exosome secretion from single cells reveals a modulatory role for GPCR signaling

    doi: 10.1083/jcb.201703206

    Figure Lengend Snippet: GPCR activation triggers MVB–PM fusion in HeLa cells via SNAP23-Ser110 phosphorylation. (a) Fusion activity of histamine-stimulated cells nontreated or treated with the Gα q inhibitor UBO-QIC (1 µM). n ≥ 24 cells per condition. (b) Basal fusion activity in cells treated with PKC inhibitors GÖ6976 (1 µM) or GÖ6983 (1 µM). n ≥ 11 cells per condition. (c) Fusion activity of histamine-stimulated cells nontreated or preincubated with GÖ6983 (1 µM). n ≥ 11 cells per condition. (d) Schematic representation of SNAP23 with SNARE motifs, a membrane-anchoring domain (M), and all phosphosites with the posphosite targeted by histamine stimulation (Ser110) in bold. (e) Fusion activity of histamine-stimulated cells transfected with WT SNAP23, phosphomutant SNAP23-S110A, or phosphomimic SNAP23-S110D. n ≥ 16 cells per condition. (f) Left: fusion activity of CD63-, CD81-, and CD9-pHluorin HeLa cells cotransfected with SNAP23 WT or SNAP23-S110A. n ≥ 16 cells per condition. Western blot on exosomes isolated from SNAP23-WT and SNAP23-S110A HeLa cells labeled for CD63, CD9, CD81, flotillin-1, and syntenin-1. (g) Schematic representation of the histamine-stimulated pathway leading to exosome release as identified by phosphoproteomics and specific inhibitors. Blue-rimmed proteins represent the putative pathway implicated by both experiments. The IP3–Ca 2+ pathway is represented in gray as a direct link with MVB–PM fusion is missing. **, P

    Article Snippet: Syntenin-1 (rabbit; 610821; BD) and flotillin-1 (rabbit; ab19903; Abcam) mouse antibodies were used at 1:250.

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

    Dsg3 and other desmosomal proteins are membrane raft associated. Primary human keratinocytes were grown to confluence and switched to high calcium media for 16–18 hrs. Following detergent extraction (1% Triton X-100) and ultra-centrifugation on a 5–40% sucrose gradient, 12 fractions were sequentially removed from the gradient and processed via western blot. Dsg3 partitions to the buoyant raft fractions (DRMs, detergent resistant membranes) as indicated by the positive controls flotillin-1 and caveolin-1, and negative control calnexin. Desmosomal components plakoglobin (PG) and plakophilin 2 (pkp-2) were also found to be raft associated. E-cadherin, a classical cadherin of adherens junctions, is not enriched in membrane rafts.

    Journal: PLoS ONE

    Article Title: Desmosome Assembly and Disassembly Are Membrane Raft-Dependent

    doi: 10.1371/journal.pone.0087809

    Figure Lengend Snippet: Dsg3 and other desmosomal proteins are membrane raft associated. Primary human keratinocytes were grown to confluence and switched to high calcium media for 16–18 hrs. Following detergent extraction (1% Triton X-100) and ultra-centrifugation on a 5–40% sucrose gradient, 12 fractions were sequentially removed from the gradient and processed via western blot. Dsg3 partitions to the buoyant raft fractions (DRMs, detergent resistant membranes) as indicated by the positive controls flotillin-1 and caveolin-1, and negative control calnexin. Desmosomal components plakoglobin (PG) and plakophilin 2 (pkp-2) were also found to be raft associated. E-cadherin, a classical cadherin of adherens junctions, is not enriched in membrane rafts.

    Article Snippet: Antibodies and Reagents Antibodies used were as follows: mouse anti-Dsg3 antibodies AK15 and AK23 were kind gifts from Dr. Masayuki Amagai (Keio University, Tokyo); mouse anti-Dsg3 antibody 5G11 (Invitrogen, Carlsbad, CA); mouse anti-plakophilin 2 antibody (Biodesign, Saco, Maine); rabbit anti-calnexin antibody (Enzo Life Sciences, Farmingdale, NY); mouse anti-CD59-FITC conjugated antibody (Millipore, Billerica, MA; Invitrogen); rabbit anti-desmoplakin antibody NW6 was a kind gift from Dr. Kathleen Green (Northwestern University); rabbit anti-gamma catenin antibody (plakoglobin, H-80, Santa Cruz Biotechnology, Santa Cruz, CA); mouse anti-E-cadherin antibody, mouse anti-flotillin-1, rabbit anti-caveolin-1, and mouse anti-clathrin antibodies (BD Biosciences, San Jose, CA).

    Techniques: Centrifugation, Western Blot, Negative Control

    Dsg3 raft association increases upon calcium addition. ( A ) Dsg3 and DP (desmoplakin) remain cytoplasmic when human keratinocytes are cultured in low (50 µM) calcium media. Protein staining increases at regions of cell contact when keratinocytes are cultured in high (550 µM) calcium media and desmosomes assemble. Dsg3 was detected with AK23 mAb post fixation. ( B ) Confluent keratinocytes cultured in 50 µM or 550 µM calcium media 16–18 hrs prior to solubilization with 1% Triton X-100 and membrane raft fractionation. Western blots were probed for Dsg3 and the raft marker flotillin-1. Dsg3 raft partitioning increases significantly upon shifting cells from low to high calcium conditions. ( C ) Quantification of relative Dsg3 levels normalized to total Dsg3 across all fractions. Means ± SEM ( n = 3); *p

    Journal: PLoS ONE

    Article Title: Desmosome Assembly and Disassembly Are Membrane Raft-Dependent

    doi: 10.1371/journal.pone.0087809

    Figure Lengend Snippet: Dsg3 raft association increases upon calcium addition. ( A ) Dsg3 and DP (desmoplakin) remain cytoplasmic when human keratinocytes are cultured in low (50 µM) calcium media. Protein staining increases at regions of cell contact when keratinocytes are cultured in high (550 µM) calcium media and desmosomes assemble. Dsg3 was detected with AK23 mAb post fixation. ( B ) Confluent keratinocytes cultured in 50 µM or 550 µM calcium media 16–18 hrs prior to solubilization with 1% Triton X-100 and membrane raft fractionation. Western blots were probed for Dsg3 and the raft marker flotillin-1. Dsg3 raft partitioning increases significantly upon shifting cells from low to high calcium conditions. ( C ) Quantification of relative Dsg3 levels normalized to total Dsg3 across all fractions. Means ± SEM ( n = 3); *p

    Article Snippet: Antibodies and Reagents Antibodies used were as follows: mouse anti-Dsg3 antibodies AK15 and AK23 were kind gifts from Dr. Masayuki Amagai (Keio University, Tokyo); mouse anti-Dsg3 antibody 5G11 (Invitrogen, Carlsbad, CA); mouse anti-plakophilin 2 antibody (Biodesign, Saco, Maine); rabbit anti-calnexin antibody (Enzo Life Sciences, Farmingdale, NY); mouse anti-CD59-FITC conjugated antibody (Millipore, Billerica, MA; Invitrogen); rabbit anti-desmoplakin antibody NW6 was a kind gift from Dr. Kathleen Green (Northwestern University); rabbit anti-gamma catenin antibody (plakoglobin, H-80, Santa Cruz Biotechnology, Santa Cruz, CA); mouse anti-E-cadherin antibody, mouse anti-flotillin-1, rabbit anti-caveolin-1, and mouse anti-clathrin antibodies (BD Biosciences, San Jose, CA).

    Techniques: Cell Culture, Staining, Fractionation, Western Blot, Marker

    Characterization of exosomes ( A ) Representative electron microscopy image of exosomes. ( B ) Western blot analysis of CD63, CD9 and β-actin in exosomes and CNE-2 cells. Flotillin-1 was used as a loading control. ( C ) The number of exosomes was counted in 10 fields under TEM (transmission electron microscope).

    Journal: Oncotarget

    Article Title: Clinical and biological significance of HAX-1 overexpression in nasopharyngeal carcinoma

    doi: 10.18632/oncotarget.7274

    Figure Lengend Snippet: Characterization of exosomes ( A ) Representative electron microscopy image of exosomes. ( B ) Western blot analysis of CD63, CD9 and β-actin in exosomes and CNE-2 cells. Flotillin-1 was used as a loading control. ( C ) The number of exosomes was counted in 10 fields under TEM (transmission electron microscope).

    Article Snippet: Western blot analysis For western blot analysis, we used the following antibodies: anti–HAX-1 (1:500, Santa Cruz Biotechnology, CA, USA), anti–p-AKT (1:1000, Santa Cruz Biotechnology), anti–AKT (1:1000, Santa Cruz Biotechnology), anti–Flotillin-1 (1:1000, Santa Cruz Biotechnology), anti–CD63 (1:1000, Sangon Biotech, Shanghai, China), and anti–CD9 (1:1000, Sangon Biotech, Shanghai, China), as well as anti-β-actin (1:2000, Santa Cruz Biotechnology) as a loading control.

    Techniques: Electron Microscopy, Western Blot, Transmission Electron Microscopy, Transmission Assay, Microscopy

    NPC-exosomes are enriched in HAX-1 which increases proliferation, migration and angiogenesis in HUVECs ( A – B ) Western blot analysis of HAX-1 expression in exosomes isolated from NPC and normal people. Flotillin-1 was used as a loading control. ( C ) Uptake of PKH67-labeled exosomes by HUVECs. In the confocal microscopy image, Hoechst staining was used to detect nuclei of cells (blue) and PKH67 was used to label the exosomes (green). ( D ) Proliferation analysis was performed after exosome addition to HUVECs. ( E – F ) Cell migration analysis by transwell assays. Each time point was derived from three independent experiments. ( G – H ) Apoptosis rates were measured by FCM analysis after Annexin V/PI staining. ( I – J ) Representative micrographs of capillary-like structure formation on Matrigel by HUVECs unstimulated (Control) and stimulated with exosomes isolated from NPC and normal people. * P

    Journal: Oncotarget

    Article Title: Clinical and biological significance of HAX-1 overexpression in nasopharyngeal carcinoma

    doi: 10.18632/oncotarget.7274

    Figure Lengend Snippet: NPC-exosomes are enriched in HAX-1 which increases proliferation, migration and angiogenesis in HUVECs ( A – B ) Western blot analysis of HAX-1 expression in exosomes isolated from NPC and normal people. Flotillin-1 was used as a loading control. ( C ) Uptake of PKH67-labeled exosomes by HUVECs. In the confocal microscopy image, Hoechst staining was used to detect nuclei of cells (blue) and PKH67 was used to label the exosomes (green). ( D ) Proliferation analysis was performed after exosome addition to HUVECs. ( E – F ) Cell migration analysis by transwell assays. Each time point was derived from three independent experiments. ( G – H ) Apoptosis rates were measured by FCM analysis after Annexin V/PI staining. ( I – J ) Representative micrographs of capillary-like structure formation on Matrigel by HUVECs unstimulated (Control) and stimulated with exosomes isolated from NPC and normal people. * P

    Article Snippet: Western blot analysis For western blot analysis, we used the following antibodies: anti–HAX-1 (1:500, Santa Cruz Biotechnology, CA, USA), anti–p-AKT (1:1000, Santa Cruz Biotechnology), anti–AKT (1:1000, Santa Cruz Biotechnology), anti–Flotillin-1 (1:1000, Santa Cruz Biotechnology), anti–CD63 (1:1000, Sangon Biotech, Shanghai, China), and anti–CD9 (1:1000, Sangon Biotech, Shanghai, China), as well as anti-β-actin (1:2000, Santa Cruz Biotechnology) as a loading control.

    Techniques: Migration, Western Blot, Expressing, Isolation, Labeling, Confocal Microscopy, Staining, Derivative Assay

    Transfer of exosomal HAX-1 increases proliferation, migration and angiogenesis effects in HUVECs ( A – B ) Western blot analysis of HAX-1 expression in exosomes isolated from culture media of HAX_siR2, NC_siR, control, pGV-HAX-1 and mock CNE-2 cells. Flotillin-1 was used as a loading control. ( C ) HUVECs proliferation was measured after treating with exosomes containing different levels of HAX-1 protein. ( D – E ) Cell migration analysis by transwell assays. ( F – G ) Representative micrographs of capillary-like structure formation on Matrigel. * P

    Journal: Oncotarget

    Article Title: Clinical and biological significance of HAX-1 overexpression in nasopharyngeal carcinoma

    doi: 10.18632/oncotarget.7274

    Figure Lengend Snippet: Transfer of exosomal HAX-1 increases proliferation, migration and angiogenesis effects in HUVECs ( A – B ) Western blot analysis of HAX-1 expression in exosomes isolated from culture media of HAX_siR2, NC_siR, control, pGV-HAX-1 and mock CNE-2 cells. Flotillin-1 was used as a loading control. ( C ) HUVECs proliferation was measured after treating with exosomes containing different levels of HAX-1 protein. ( D – E ) Cell migration analysis by transwell assays. ( F – G ) Representative micrographs of capillary-like structure formation on Matrigel. * P

    Article Snippet: Western blot analysis For western blot analysis, we used the following antibodies: anti–HAX-1 (1:500, Santa Cruz Biotechnology, CA, USA), anti–p-AKT (1:1000, Santa Cruz Biotechnology), anti–AKT (1:1000, Santa Cruz Biotechnology), anti–Flotillin-1 (1:1000, Santa Cruz Biotechnology), anti–CD63 (1:1000, Sangon Biotech, Shanghai, China), and anti–CD9 (1:1000, Sangon Biotech, Shanghai, China), as well as anti-β-actin (1:2000, Santa Cruz Biotechnology) as a loading control.

    Techniques: Migration, Western Blot, Expressing, Isolation

    Distribution of ABCA1, ABCG1, and ABCG4 between Triton X-100 raft and non-raft domains. HEK293, HEK/ABCA1, HEK/ABCG1, HEK/ABCG1-KM, or HEK/ABCG4 cells (A) or SH-SY5Y cells (B) treated with TO901317 and 9-cis retinoic acid for 24 h were incubated with buffer containing 1% Triton X-100 on ice. The cell lysates were separated by OptiPrep-gradient ultracentrifugation. Ten fractions of each were separated by 5–20% polyacrylamide gel electrophoresis, and ABCA1, ABCG1, ABCG4, caveolin-1, paxillin, FAK, or flotillin was detected by immunoblotting.

    Journal: PLoS ONE

    Article Title: ABCA1, ABCG1, and ABCG4 Are Distributed to Distinct Membrane Meso-Domains and Disturb Detergent-Resistant Domains on the Plasma Membrane

    doi: 10.1371/journal.pone.0109886

    Figure Lengend Snippet: Distribution of ABCA1, ABCG1, and ABCG4 between Triton X-100 raft and non-raft domains. HEK293, HEK/ABCA1, HEK/ABCG1, HEK/ABCG1-KM, or HEK/ABCG4 cells (A) or SH-SY5Y cells (B) treated with TO901317 and 9-cis retinoic acid for 24 h were incubated with buffer containing 1% Triton X-100 on ice. The cell lysates were separated by OptiPrep-gradient ultracentrifugation. Ten fractions of each were separated by 5–20% polyacrylamide gel electrophoresis, and ABCA1, ABCG1, ABCG4, caveolin-1, paxillin, FAK, or flotillin was detected by immunoblotting.

    Article Snippet: Materials Rabbit polyclonal anti-ABCG1 and anti-flotillin-1 antibodies were purchased from Santa Cruz (Santa Cruz, CA).

    Techniques: Incubation, Polyacrylamide Gel Electrophoresis

    Colocalization of ABCG1 with flotillin-1. HEK293 cells transiently expressing ABCG1, ABCG1-KM, ABCG4, or ABCG4-KM plus flotillin-GFP were permeabilized with Triton X-100 (ABCG1) or methanol (ABCG4) and reacted with anti-ABCG1 or -ABCG4 antibodies and analyzed by confocal microscopy. Line scans analyzed by ImageJ software indicate the fluorescence intensities of flotillin-1 (green), ABCG1 (red), and ABCG4 (red).

    Journal: PLoS ONE

    Article Title: ABCA1, ABCG1, and ABCG4 Are Distributed to Distinct Membrane Meso-Domains and Disturb Detergent-Resistant Domains on the Plasma Membrane

    doi: 10.1371/journal.pone.0109886

    Figure Lengend Snippet: Colocalization of ABCG1 with flotillin-1. HEK293 cells transiently expressing ABCG1, ABCG1-KM, ABCG4, or ABCG4-KM plus flotillin-GFP were permeabilized with Triton X-100 (ABCG1) or methanol (ABCG4) and reacted with anti-ABCG1 or -ABCG4 antibodies and analyzed by confocal microscopy. Line scans analyzed by ImageJ software indicate the fluorescence intensities of flotillin-1 (green), ABCG1 (red), and ABCG4 (red).

    Article Snippet: Materials Rabbit polyclonal anti-ABCG1 and anti-flotillin-1 antibodies were purchased from Santa Cruz (Santa Cruz, CA).

    Techniques: Expressing, Confocal Microscopy, Software, Fluorescence

    Non-raft association of PS1 and APP CTFs in embryonic mouse brain Raft and non-raft distribution of PS1, nicastrin, APP, flotillin-2, calnexin, and γ-adaptin in flotation density gradient fractions from E15.5 embryonic brains were analyzed by Western blotting. Note the predominant non-raft localization of PS1, nicastrin, and APP CTFs. mat , mature; imm , immature.

    Journal: The Journal of biological chemistry

    Article Title: Spatial Segregation of ?-Secretase and Substrates in Distinct Membrane Domains *

    doi: 10.1074/jbc.M503570200

    Figure Lengend Snippet: Non-raft association of PS1 and APP CTFs in embryonic mouse brain Raft and non-raft distribution of PS1, nicastrin, APP, flotillin-2, calnexin, and γ-adaptin in flotation density gradient fractions from E15.5 embryonic brains were analyzed by Western blotting. Note the predominant non-raft localization of PS1, nicastrin, and APP CTFs. mat , mature; imm , immature.

    Article Snippet: The following antibodies were purchased from commercial sources: flotillin-2, syntaxin 6, γ-adaptin, Notch1, N-cadherin, and DCC were from BD Transduction Laboratories; OKT8 and 9E10 were from the ATCC; caveolin-1 was from Santa Cruz Biotechnology; and calnexin was from Stressgen.

    Techniques: Western Blot

    DIM association of BACE-1, γ-secretase, and APP CTFs in mouse brain 12-Month-old adult mouse brain was homogenized and solubilized in a buffer containing 0.5% Lubrol WX at 4 °C for 30 min. The lysates were then subject to flotation sucrose density gradient centrifugation as described under “Experimental Procedures.” The gradients were harvested from the top, and equal volume of each fraction was analyzed by Western blotting with antibodies against APP C terminus, BACE-1, PS1, nicastrin, and PEN-2. Fractions 4 and 5 represent the interface between 5 and 35% sucrose in the gradient and are enriched in lipid raft marker flotillin-2. Fractions 8–12 contain detergent-soluble proteins, marked by the presence of non-raft proteins γ-adaptin and calnexin.

    Journal: The Journal of biological chemistry

    Article Title: Spatial Segregation of ?-Secretase and Substrates in Distinct Membrane Domains *

    doi: 10.1074/jbc.M503570200

    Figure Lengend Snippet: DIM association of BACE-1, γ-secretase, and APP CTFs in mouse brain 12-Month-old adult mouse brain was homogenized and solubilized in a buffer containing 0.5% Lubrol WX at 4 °C for 30 min. The lysates were then subject to flotation sucrose density gradient centrifugation as described under “Experimental Procedures.” The gradients were harvested from the top, and equal volume of each fraction was analyzed by Western blotting with antibodies against APP C terminus, BACE-1, PS1, nicastrin, and PEN-2. Fractions 4 and 5 represent the interface between 5 and 35% sucrose in the gradient and are enriched in lipid raft marker flotillin-2. Fractions 8–12 contain detergent-soluble proteins, marked by the presence of non-raft proteins γ-adaptin and calnexin.

    Article Snippet: The following antibodies were purchased from commercial sources: flotillin-2, syntaxin 6, γ-adaptin, Notch1, N-cadherin, and DCC were from BD Transduction Laboratories; OKT8 and 9E10 were from the ATCC; caveolin-1 was from Santa Cruz Biotechnology; and calnexin was from Stressgen.

    Techniques: Gradient Centrifugation, Western Blot, Marker

    DIM accumulation of APP CTFs in cells lacking γ-secretase activity wt and PS1 −/− /PS2 −/− fibroblasts were solubilized in 0.5% Lubrol WX and analyzed by sucrose gradient fractionation and Western blotting with antibodies against APP, flotillin-2, and cavelolin-1. In the absence of PS1 and PS2 expression, endogenous wt APP CTFs become Lubrol WX-resistant and were recovered mainly in raft fractions 4 and 5, identified by the presence of raft markers.

    Journal: The Journal of biological chemistry

    Article Title: Spatial Segregation of ?-Secretase and Substrates in Distinct Membrane Domains *

    doi: 10.1074/jbc.M503570200

    Figure Lengend Snippet: DIM accumulation of APP CTFs in cells lacking γ-secretase activity wt and PS1 −/− /PS2 −/− fibroblasts were solubilized in 0.5% Lubrol WX and analyzed by sucrose gradient fractionation and Western blotting with antibodies against APP, flotillin-2, and cavelolin-1. In the absence of PS1 and PS2 expression, endogenous wt APP CTFs become Lubrol WX-resistant and were recovered mainly in raft fractions 4 and 5, identified by the presence of raft markers.

    Article Snippet: The following antibodies were purchased from commercial sources: flotillin-2, syntaxin 6, γ-adaptin, Notch1, N-cadherin, and DCC were from BD Transduction Laboratories; OKT8 and 9E10 were from the ATCC; caveolin-1 was from Santa Cruz Biotechnology; and calnexin was from Stressgen.

    Techniques: Activity Assay, Fractionation, Western Blot, Expressing

    Covalent labeling of PS1 by active site directed 3-secretase inhibitor L-852,505 Pooled raft and non-raft fractions from wt mouse embryonic fibroblasts ( A ), N2a wt.11 cells ( B ), or adult mouse brain ( C ) were incubated with Me 2 SO (− inhibitor ) or L-852,505 (+ inhibitor ), followed by irradiation with 365 nm UV light on ice for 90 min to allow photoaffinity cross-linking. Biotinylated proteins were captured with streptavidin-agarose beads and eluted by incubation at 95 °C for 5 min in Laemmli/SDS sample buffer. The eluates ( lanes 5–9 ) as well as 1/40th of the solubilized membranes used for the streptavidin ( Strep. ) precipitation ( input ) were analyzed by immunoblotting using αPS1Loop antibody. The left panels show the distribution of PS1 and flotillin-2 in the flotation density gradient fractions used for the in vitro cross-linking experiment. IP , immunoprecipitation.

    Journal: The Journal of biological chemistry

    Article Title: Spatial Segregation of ?-Secretase and Substrates in Distinct Membrane Domains *

    doi: 10.1074/jbc.M503570200

    Figure Lengend Snippet: Covalent labeling of PS1 by active site directed 3-secretase inhibitor L-852,505 Pooled raft and non-raft fractions from wt mouse embryonic fibroblasts ( A ), N2a wt.11 cells ( B ), or adult mouse brain ( C ) were incubated with Me 2 SO (− inhibitor ) or L-852,505 (+ inhibitor ), followed by irradiation with 365 nm UV light on ice for 90 min to allow photoaffinity cross-linking. Biotinylated proteins were captured with streptavidin-agarose beads and eluted by incubation at 95 °C for 5 min in Laemmli/SDS sample buffer. The eluates ( lanes 5–9 ) as well as 1/40th of the solubilized membranes used for the streptavidin ( Strep. ) precipitation ( input ) were analyzed by immunoblotting using αPS1Loop antibody. The left panels show the distribution of PS1 and flotillin-2 in the flotation density gradient fractions used for the in vitro cross-linking experiment. IP , immunoprecipitation.

    Article Snippet: The following antibodies were purchased from commercial sources: flotillin-2, syntaxin 6, γ-adaptin, Notch1, N-cadherin, and DCC were from BD Transduction Laboratories; OKT8 and 9E10 were from the ATCC; caveolin-1 was from Santa Cruz Biotechnology; and calnexin was from Stressgen.

    Techniques: Labeling, Incubation, Irradiation, In Vitro, Immunoprecipitation

    Localization and cytotoxicity of VVH are not affected by SMase. CHO cells were incubated with (+) or without (−) 100 mU/ml SMase at 37°C for 1 h. After incubation, the cells were washed twice with DMEM and incubated with 5 µg/ml VVH at 37°C for 15 min. Cells were lysed, and fractionated. VVH, flotillin-1 (flt-1), and transferrin receptor (TfR) were detected by western blotting using specific antibodies.

    Journal: PLoS ONE

    Article Title: Relationship between Localization on Cellular Membranes and Cytotoxicity of Vibrio vulnificus Hemolysin

    doi: 10.1371/journal.pone.0026018

    Figure Lengend Snippet: Localization and cytotoxicity of VVH are not affected by SMase. CHO cells were incubated with (+) or without (−) 100 mU/ml SMase at 37°C for 1 h. After incubation, the cells were washed twice with DMEM and incubated with 5 µg/ml VVH at 37°C for 15 min. Cells were lysed, and fractionated. VVH, flotillin-1 (flt-1), and transferrin receptor (TfR) were detected by western blotting using specific antibodies.

    Article Snippet: The presence of actin, flotillin-1, transferrin receptor (TfR), caveoline-1 and VVH were detected using anti-actin monoclonal antibody clone C4 (Chemicon International Inc., Temecula, CA ), anti-flotillin-1 monoclonal antibody clone 18 (BD Biosciences, San Jose, CA), anti-TfR monoclonal antibody clone H68.4 (Zymed Laboratories Inc., South San Francisco, CA), anti-caveolin-1 monoclonal antibody clone 2297 (BD Biosciences, San Jose, CA) and anti-VVH polyclonal antibody, respectively.

    Techniques: Incubation, Western Blot

    MβCD changes the localization of VVH. CHO cells were incubated with (+) or without (−) 8 mM MβCD at 37°C for 1 h. After incubation, the cells were washed twice with DMEM and incubated with 5 µg/ml VVH at 37°C for 15 min. Cells were lysed, and fractionated. VVH, flotillin-1 (flt-1), and transferrin receptor (TfR) were detected by western blotting using specific antibodies.

    Journal: PLoS ONE

    Article Title: Relationship between Localization on Cellular Membranes and Cytotoxicity of Vibrio vulnificus Hemolysin

    doi: 10.1371/journal.pone.0026018

    Figure Lengend Snippet: MβCD changes the localization of VVH. CHO cells were incubated with (+) or without (−) 8 mM MβCD at 37°C for 1 h. After incubation, the cells were washed twice with DMEM and incubated with 5 µg/ml VVH at 37°C for 15 min. Cells were lysed, and fractionated. VVH, flotillin-1 (flt-1), and transferrin receptor (TfR) were detected by western blotting using specific antibodies.

    Article Snippet: The presence of actin, flotillin-1, transferrin receptor (TfR), caveoline-1 and VVH were detected using anti-actin monoclonal antibody clone C4 (Chemicon International Inc., Temecula, CA ), anti-flotillin-1 monoclonal antibody clone 18 (BD Biosciences, San Jose, CA), anti-TfR monoclonal antibody clone H68.4 (Zymed Laboratories Inc., South San Francisco, CA), anti-caveolin-1 monoclonal antibody clone 2297 (BD Biosciences, San Jose, CA) and anti-VVH polyclonal antibody, respectively.

    Techniques: Incubation, Western Blot

    VVH associates with DRMs. CHO cells were incubated with 5 µg/ml VVH at 37°C for 15 min. After being fractionated by sucrose gradient ultracentrifugation, VVH, flotillin-1 (flt-1), and transferrin receptor (TfR) were detected by western blotting using specific antibodies.

    Journal: PLoS ONE

    Article Title: Relationship between Localization on Cellular Membranes and Cytotoxicity of Vibrio vulnificus Hemolysin

    doi: 10.1371/journal.pone.0026018

    Figure Lengend Snippet: VVH associates with DRMs. CHO cells were incubated with 5 µg/ml VVH at 37°C for 15 min. After being fractionated by sucrose gradient ultracentrifugation, VVH, flotillin-1 (flt-1), and transferrin receptor (TfR) were detected by western blotting using specific antibodies.

    Article Snippet: The presence of actin, flotillin-1, transferrin receptor (TfR), caveoline-1 and VVH were detected using anti-actin monoclonal antibody clone C4 (Chemicon International Inc., Temecula, CA ), anti-flotillin-1 monoclonal antibody clone 18 (BD Biosciences, San Jose, CA), anti-TfR monoclonal antibody clone H68.4 (Zymed Laboratories Inc., South San Francisco, CA), anti-caveolin-1 monoclonal antibody clone 2297 (BD Biosciences, San Jose, CA) and anti-VVH polyclonal antibody, respectively.

    Techniques: Incubation, Western Blot

    Effect of cholesterol depletion on SNARE protein association with lipid rafts. ( A , B ) Isolation of lipid rafts from alveolar type II cells. Lipid rafts were isolated from freshly isolated type II cells based on their detergent insolubility in 1% Triton X-100 floatation on sucrose gradients. Seven fractions (1–7) were collected from top to bottom and the pellet solubilized in lysis buffer (fraction 8). Protein ( A ) and cholesterol ( B ) were measured in each of the fractions before and after cholesterol depletion (0.5% Triton X-100 and 0.5%, wt/vol saponin). The results were expressed as a percent of total protein and cholesterol, respectively. Data shown are means ± SE ( n = 3). Diamonds , 1% Triton X-100; circles , 0.5% Triton X-100 and 0.5% saponin. ( C ) SNARE association with lipid rafts. Type II cells were lysed in either 1% Triton X-100 (control) or 0.5% Triton X-100 and 0.5% saponin (cholesterol depletion) and subjected to raft isolation. Seven fractions were collected from top to bottom. P, pellet. Percentages (5, 30, and 40%) represent sucrose gradient. Each fraction was examined by Western blot for the presence of the lipid raft marker proteins flotillin-1 and -2, the non–lipid raft protein marker Na + -K + ATPase, various SNARE proteins, SNAP-23, syntaxin 2, and VAMP-2. The immunublots shown are representative of three independent experiments.

    Journal: American Journal of Respiratory Cell and Molecular Biology

    Article Title: Effect of Cholesterol Depletion on Exocytosis of Alveolar Type II Cells

    doi: 10.1165/rcmb.2005-0418OC

    Figure Lengend Snippet: Effect of cholesterol depletion on SNARE protein association with lipid rafts. ( A , B ) Isolation of lipid rafts from alveolar type II cells. Lipid rafts were isolated from freshly isolated type II cells based on their detergent insolubility in 1% Triton X-100 floatation on sucrose gradients. Seven fractions (1–7) were collected from top to bottom and the pellet solubilized in lysis buffer (fraction 8). Protein ( A ) and cholesterol ( B ) were measured in each of the fractions before and after cholesterol depletion (0.5% Triton X-100 and 0.5%, wt/vol saponin). The results were expressed as a percent of total protein and cholesterol, respectively. Data shown are means ± SE ( n = 3). Diamonds , 1% Triton X-100; circles , 0.5% Triton X-100 and 0.5% saponin. ( C ) SNARE association with lipid rafts. Type II cells were lysed in either 1% Triton X-100 (control) or 0.5% Triton X-100 and 0.5% saponin (cholesterol depletion) and subjected to raft isolation. Seven fractions were collected from top to bottom. P, pellet. Percentages (5, 30, and 40%) represent sucrose gradient. Each fraction was examined by Western blot for the presence of the lipid raft marker proteins flotillin-1 and -2, the non–lipid raft protein marker Na + -K + ATPase, various SNARE proteins, SNAP-23, syntaxin 2, and VAMP-2. The immunublots shown are representative of three independent experiments.

    Article Snippet: Monoclonal anti-flotillin–1 and –2, anti–α-SNAP, anti-NSF, and anti-CD44 antibodies were from BD Transduction Laboratories (Lexington, KY).

    Techniques: Isolation, Lysis, Western Blot, Marker

    Identification of lipid raft markers in alveolar type II cells. ( A ) RT-PCR. The mRNAs extracted from freshly isolated alveolar type II cells ( > 96% purity, T2) and whole lung tissue (L) were reverse-transcribed and amplified for 35 cycles with gene specific primers against caveolin (Cav) 1α, 1β, 2, and 3, flotillin (Flot) 1 and 2, and 18S rRNA. PCR products were electrophoretically separated on an agarose gel. MW: 100-bp DNA ladder. ( B ) Western blot. Freshly isolated type II cells (D0), 3- or 7-d cultured type II cells on plastic dishes (D3 and D7), lung epithelial cell lines, MLE-12 and L2 cells, along with lung homogenate (LH), purified plasma membrane (PM), and lamellar bodies (LB), were either lysed and/or solubilized in SDS sample buffer. Equal amounts of total protein (20 μg) were separated by SDS-PAGE and immunoblotted using anti–flotillin-1 and -2 antibodies. ( C ) Immunohistochemistry. Lung tissue sections were permeabilized and blocked before being incubated with goat anti–SP-C and mouse anti–flotillin-1 and -2 antibodies. They were then incubated with donkey anti-goat Alexa 546–conjugated and rabbit anti-mouse Alexa 488–conjugated antibodies. The slides were observed for immunoflorescence. Upper panels : double labeling with anti–flotillin-1 and SP-C antibodies; lower panels : double labeling with anti–flotillin-2 and SP-C antibodies. Shown in the inset is the magnified image (×100) of a single type II cell. Scale bar : 50 μm.

    Journal: American Journal of Respiratory Cell and Molecular Biology

    Article Title: Effect of Cholesterol Depletion on Exocytosis of Alveolar Type II Cells

    doi: 10.1165/rcmb.2005-0418OC

    Figure Lengend Snippet: Identification of lipid raft markers in alveolar type II cells. ( A ) RT-PCR. The mRNAs extracted from freshly isolated alveolar type II cells ( > 96% purity, T2) and whole lung tissue (L) were reverse-transcribed and amplified for 35 cycles with gene specific primers against caveolin (Cav) 1α, 1β, 2, and 3, flotillin (Flot) 1 and 2, and 18S rRNA. PCR products were electrophoretically separated on an agarose gel. MW: 100-bp DNA ladder. ( B ) Western blot. Freshly isolated type II cells (D0), 3- or 7-d cultured type II cells on plastic dishes (D3 and D7), lung epithelial cell lines, MLE-12 and L2 cells, along with lung homogenate (LH), purified plasma membrane (PM), and lamellar bodies (LB), were either lysed and/or solubilized in SDS sample buffer. Equal amounts of total protein (20 μg) were separated by SDS-PAGE and immunoblotted using anti–flotillin-1 and -2 antibodies. ( C ) Immunohistochemistry. Lung tissue sections were permeabilized and blocked before being incubated with goat anti–SP-C and mouse anti–flotillin-1 and -2 antibodies. They were then incubated with donkey anti-goat Alexa 546–conjugated and rabbit anti-mouse Alexa 488–conjugated antibodies. The slides were observed for immunoflorescence. Upper panels : double labeling with anti–flotillin-1 and SP-C antibodies; lower panels : double labeling with anti–flotillin-2 and SP-C antibodies. Shown in the inset is the magnified image (×100) of a single type II cell. Scale bar : 50 μm.

    Article Snippet: Monoclonal anti-flotillin–1 and –2, anti–α-SNAP, anti-NSF, and anti-CD44 antibodies were from BD Transduction Laboratories (Lexington, KY).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Isolation, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Western Blot, Cell Culture, Purification, SDS Page, Immunohistochemistry, Incubation, Labeling

    Flotillin-2 is found in pre-fusion myoblasts after cholesterol depletion. Immunofluorescence of flotillin-2, desmin and DAPI in cultures treated with methyl-β-cyclodextrin (MbCD) showing flotillin-2 expression in mononucleated cells that are fusing with myotubes. Chick myogenic cells were grown for 24 hours, treated with 2 mM MbCD for 30 min and grown for the next 24 hours ( B and C ). Some cells were not treated and were fixed at 48 hours of culture (control, A ). All cells were fixed with paraformaldehyde and stained with antibodies against desmin ( red ) and flotillin-2 ( green ) and with the nuclear dye DAPI ( blue ). Merged images are shown in A–C . Note an increase in the number of flotillin-2 positive-mononucleated cells in close contact with the membrane of multinucleated myotubes (arrows in B and C ). Scale bar in A represents 20 µm.

    Journal: PLoS ONE

    Article Title: Differences in the Expression and Distribution of Flotillin-2 in Chick, Mice and Human Muscle Cells

    doi: 10.1371/journal.pone.0103990

    Figure Lengend Snippet: Flotillin-2 is found in pre-fusion myoblasts after cholesterol depletion. Immunofluorescence of flotillin-2, desmin and DAPI in cultures treated with methyl-β-cyclodextrin (MbCD) showing flotillin-2 expression in mononucleated cells that are fusing with myotubes. Chick myogenic cells were grown for 24 hours, treated with 2 mM MbCD for 30 min and grown for the next 24 hours ( B and C ). Some cells were not treated and were fixed at 48 hours of culture (control, A ). All cells were fixed with paraformaldehyde and stained with antibodies against desmin ( red ) and flotillin-2 ( green ) and with the nuclear dye DAPI ( blue ). Merged images are shown in A–C . Note an increase in the number of flotillin-2 positive-mononucleated cells in close contact with the membrane of multinucleated myotubes (arrows in B and C ). Scale bar in A represents 20 µm.

    Article Snippet: The sections were blocked in 50 mM NH4 Cl and 3% bovine serum albumin in PBS, pH 8.0 for 30 min, and incubated with a monoclonal anti-flotillin-2 antibody (BD Transduction Laboratories, USA) diluted at 1∶5, followed by a goat anti-mouse 10-η m gold-conjugated (BB International, USA) diluted at 1∶100.

    Techniques: Immunofluorescence, Expressing, Staining

    Distribution of flotillin-2 in human neonatal muscle cells. Human neonatal muscle cells were grown as described in Materials and Methods section. Cells were fixed with methanol and stained with an anti-flotillin-2 antibody ( green , A and B ) and the nuclear dye DAPI ( blue , A and B ). Merged images are shown in A and B . Note that flotillin-2 is present in human myoblasts and myotubes in vesicle-like structures (arrows in A and B ). Scale bar in A represents 10 µm.

    Journal: PLoS ONE

    Article Title: Differences in the Expression and Distribution of Flotillin-2 in Chick, Mice and Human Muscle Cells

    doi: 10.1371/journal.pone.0103990

    Figure Lengend Snippet: Distribution of flotillin-2 in human neonatal muscle cells. Human neonatal muscle cells were grown as described in Materials and Methods section. Cells were fixed with methanol and stained with an anti-flotillin-2 antibody ( green , A and B ) and the nuclear dye DAPI ( blue , A and B ). Merged images are shown in A and B . Note that flotillin-2 is present in human myoblasts and myotubes in vesicle-like structures (arrows in A and B ). Scale bar in A represents 10 µm.

    Article Snippet: The sections were blocked in 50 mM NH4 Cl and 3% bovine serum albumin in PBS, pH 8.0 for 30 min, and incubated with a monoclonal anti-flotillin-2 antibody (BD Transduction Laboratories, USA) diluted at 1∶5, followed by a goat anti-mouse 10-η m gold-conjugated (BB International, USA) diluted at 1∶100.

    Techniques: Staining

    Cholesterol depletion enhances the expression of flotillin-2 protein and mRNA. Chick myogenic cells were grown 24(control, Ct ). Cell culture extracts were analyzed in Western blot using an antibody against flotillin-2 ( A ). Lower Western blot shows α-tubulin reactivity of the same samples, and was used to normalize sample loading ( A ). Quantification of protein bands revealed a 40% increase in the levels of flotillin-2 expression after cholesterol depletion ( B ). RT-PCR analysis (for details, see Materials and Methods ) of the expression of flotillin-2 in control and in MbCD-treated cells is shown in C . Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used for normalization. Analysis of the expression of flotillin-2 shows a more than 2-fold increase in the levels of mRNA expression in MbCD-treated cells compared with control cells. *p

    Journal: PLoS ONE

    Article Title: Differences in the Expression and Distribution of Flotillin-2 in Chick, Mice and Human Muscle Cells

    doi: 10.1371/journal.pone.0103990

    Figure Lengend Snippet: Cholesterol depletion enhances the expression of flotillin-2 protein and mRNA. Chick myogenic cells were grown 24(control, Ct ). Cell culture extracts were analyzed in Western blot using an antibody against flotillin-2 ( A ). Lower Western blot shows α-tubulin reactivity of the same samples, and was used to normalize sample loading ( A ). Quantification of protein bands revealed a 40% increase in the levels of flotillin-2 expression after cholesterol depletion ( B ). RT-PCR analysis (for details, see Materials and Methods ) of the expression of flotillin-2 in control and in MbCD-treated cells is shown in C . Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used for normalization. Analysis of the expression of flotillin-2 shows a more than 2-fold increase in the levels of mRNA expression in MbCD-treated cells compared with control cells. *p

    Article Snippet: The sections were blocked in 50 mM NH4 Cl and 3% bovine serum albumin in PBS, pH 8.0 for 30 min, and incubated with a monoclonal anti-flotillin-2 antibody (BD Transduction Laboratories, USA) diluted at 1∶5, followed by a goat anti-mouse 10-η m gold-conjugated (BB International, USA) diluted at 1∶100.

    Techniques: Expressing, Cell Culture, Western Blot, Reverse Transcription Polymerase Chain Reaction

    Cryo-immunogold EM labeling of flotillin-2 in myogenic cells. Chick myogenic cells were grown for 48-flotillin-2 antibody followed by a 10- η m gold-conjugated secondary antibody. Note that gold particles are present in vesicles ( a ) and associated with Golgi stacks ( b ). GC, Golgi complex. Scale bar in a represents 100 η m and in b represents 250 η m.

    Journal: PLoS ONE

    Article Title: Differences in the Expression and Distribution of Flotillin-2 in Chick, Mice and Human Muscle Cells

    doi: 10.1371/journal.pone.0103990

    Figure Lengend Snippet: Cryo-immunogold EM labeling of flotillin-2 in myogenic cells. Chick myogenic cells were grown for 48-flotillin-2 antibody followed by a 10- η m gold-conjugated secondary antibody. Note that gold particles are present in vesicles ( a ) and associated with Golgi stacks ( b ). GC, Golgi complex. Scale bar in a represents 100 η m and in b represents 250 η m.

    Article Snippet: The sections were blocked in 50 mM NH4 Cl and 3% bovine serum albumin in PBS, pH 8.0 for 30 min, and incubated with a monoclonal anti-flotillin-2 antibody (BD Transduction Laboratories, USA) diluted at 1∶5, followed by a goat anti-mouse 10-η m gold-conjugated (BB International, USA) diluted at 1∶100.

    Techniques: Labeling

    Brefeldin A induces a major reduction in the number of flotillin-2 containing vesicles. Chick myogenic cells were grown for 24(5 µg/ml) for 3 hours or with nocodazole (10 µg/ml) for 3 hours. After treatment, cells were grown in fresh culture medium for the next 3 hours. In images ( A–F ) cells were fixed with paraformaldehyde and stained with an antibody against flotillin-2 ( green, A,C,E ) and with the nuclear dye DAPI ( blue, B,D,F ). Note that while nocodazole have no effect in the presence and distribution of flotillin-2 positive vesicles ( C,D ), brefeldin A induced a major reduction in flotillin-2 containing vesicles ( E,F ). In images ( G–I ) cells were analyzed under phase contrast microscopy and superimposed with DAPI (blue). Scale bars represents 50 µm (in A–F and G–I ).

    Journal: PLoS ONE

    Article Title: Differences in the Expression and Distribution of Flotillin-2 in Chick, Mice and Human Muscle Cells

    doi: 10.1371/journal.pone.0103990

    Figure Lengend Snippet: Brefeldin A induces a major reduction in the number of flotillin-2 containing vesicles. Chick myogenic cells were grown for 24(5 µg/ml) for 3 hours or with nocodazole (10 µg/ml) for 3 hours. After treatment, cells were grown in fresh culture medium for the next 3 hours. In images ( A–F ) cells were fixed with paraformaldehyde and stained with an antibody against flotillin-2 ( green, A,C,E ) and with the nuclear dye DAPI ( blue, B,D,F ). Note that while nocodazole have no effect in the presence and distribution of flotillin-2 positive vesicles ( C,D ), brefeldin A induced a major reduction in flotillin-2 containing vesicles ( E,F ). In images ( G–I ) cells were analyzed under phase contrast microscopy and superimposed with DAPI (blue). Scale bars represents 50 µm (in A–F and G–I ).

    Article Snippet: The sections were blocked in 50 mM NH4 Cl and 3% bovine serum albumin in PBS, pH 8.0 for 30 min, and incubated with a monoclonal anti-flotillin-2 antibody (BD Transduction Laboratories, USA) diluted at 1∶5, followed by a goat anti-mouse 10-η m gold-conjugated (BB International, USA) diluted at 1∶100.

    Techniques: Staining, Microscopy

    Flotillin-2 is down-regulated during in vitro chick skeletal myogenesis. Chick myogenic cells were grown for 24, 48 and 72-2. ( A ) Upper Western blot shows flotillin-2 reactivity and lower Western blot shows α-tubulin reactivity of the same samples, and was used to normalize sample loading. ( B ) Quantification of protein bands revealed a progressive decrease in the levels of flotillin-2 expression during skeletal muscle differentiation. *p

    Journal: PLoS ONE

    Article Title: Differences in the Expression and Distribution of Flotillin-2 in Chick, Mice and Human Muscle Cells

    doi: 10.1371/journal.pone.0103990

    Figure Lengend Snippet: Flotillin-2 is down-regulated during in vitro chick skeletal myogenesis. Chick myogenic cells were grown for 24, 48 and 72-2. ( A ) Upper Western blot shows flotillin-2 reactivity and lower Western blot shows α-tubulin reactivity of the same samples, and was used to normalize sample loading. ( B ) Quantification of protein bands revealed a progressive decrease in the levels of flotillin-2 expression during skeletal muscle differentiation. *p

    Article Snippet: The sections were blocked in 50 mM NH4 Cl and 3% bovine serum albumin in PBS, pH 8.0 for 30 min, and incubated with a monoclonal anti-flotillin-2 antibody (BD Transduction Laboratories, USA) diluted at 1∶5, followed by a goat anti-mouse 10-η m gold-conjugated (BB International, USA) diluted at 1∶100.

    Techniques: In Vitro, Western Blot, Expressing

    Flotillin-2 is mainly expressed in fibroblasts and weakly expressed in myoblasts. Myogenic cells were grown for 24( A and B ). Cells were fixed with paraformaldehyde and stained with antibodies against MyoD ( red , A and B ) and flotillin-2 ( green , A , and B ) and with the nuclear dye DAPI ( blue , B ). Merged images are shown in A and B . Note that flotillin-2 is highly expressed in MyoD-negative cells and weakly expressed in MyoD-positive cells ( A and B ). Scale bar in B represents 20 µm.

    Journal: PLoS ONE

    Article Title: Differences in the Expression and Distribution of Flotillin-2 in Chick, Mice and Human Muscle Cells

    doi: 10.1371/journal.pone.0103990

    Figure Lengend Snippet: Flotillin-2 is mainly expressed in fibroblasts and weakly expressed in myoblasts. Myogenic cells were grown for 24( A and B ). Cells were fixed with paraformaldehyde and stained with antibodies against MyoD ( red , A and B ) and flotillin-2 ( green , A , and B ) and with the nuclear dye DAPI ( blue , B ). Merged images are shown in A and B . Note that flotillin-2 is highly expressed in MyoD-negative cells and weakly expressed in MyoD-positive cells ( A and B ). Scale bar in B represents 20 µm.

    Article Snippet: The sections were blocked in 50 mM NH4 Cl and 3% bovine serum albumin in PBS, pH 8.0 for 30 min, and incubated with a monoclonal anti-flotillin-2 antibody (BD Transduction Laboratories, USA) diluted at 1∶5, followed by a goat anti-mouse 10-η m gold-conjugated (BB International, USA) diluted at 1∶100.

    Techniques: Staining

    Flotillin-2 distribution in myogenic cells during skeletal muscle differentiation. Myogenic cells were grown for 24( A and B ) or 72 hours ( C ). Cells were fixed with paraformaldehyde and stained with antibodies against desmin ( red , A and C ) and flotillin-2 ( green , A, B and C ) and with the nuclear dye DAPI ( blue , A and C ). Merged images are shown in A and C . Note that with paraformaldehyde fixation it is possible to see flotillin-2 almost exclusively in mononucleated cells in vesicle-like structures ( B ) and nearly absent from myotubes ( A and C ). Scale bar in A and C represents 20 µm and in B represents 10 µm.

    Journal: PLoS ONE

    Article Title: Differences in the Expression and Distribution of Flotillin-2 in Chick, Mice and Human Muscle Cells

    doi: 10.1371/journal.pone.0103990

    Figure Lengend Snippet: Flotillin-2 distribution in myogenic cells during skeletal muscle differentiation. Myogenic cells were grown for 24( A and B ) or 72 hours ( C ). Cells were fixed with paraformaldehyde and stained with antibodies against desmin ( red , A and C ) and flotillin-2 ( green , A, B and C ) and with the nuclear dye DAPI ( blue , A and C ). Merged images are shown in A and C . Note that with paraformaldehyde fixation it is possible to see flotillin-2 almost exclusively in mononucleated cells in vesicle-like structures ( B ) and nearly absent from myotubes ( A and C ). Scale bar in A and C represents 20 µm and in B represents 10 µm.

    Article Snippet: The sections were blocked in 50 mM NH4 Cl and 3% bovine serum albumin in PBS, pH 8.0 for 30 min, and incubated with a monoclonal anti-flotillin-2 antibody (BD Transduction Laboratories, USA) diluted at 1∶5, followed by a goat anti-mouse 10-η m gold-conjugated (BB International, USA) diluted at 1∶100.

    Techniques: Staining

    Distribution of flotillin-2 in C2C12 mouse muscle cell line. C2C12 cells were grown as described in Materials and Methods section. Cells were fixed with methanol and stained with an anti-flotillin-2 antibody ( green , A and C ) and the nuclear dye DAPI ( blue , B and C ). A merged image is shown in C . Note that flotillin-2 is present in C2C12 cells in vesicle-like structures (arrows in A and C ). Scale bar in C represents 20 µm.

    Journal: PLoS ONE

    Article Title: Differences in the Expression and Distribution of Flotillin-2 in Chick, Mice and Human Muscle Cells

    doi: 10.1371/journal.pone.0103990

    Figure Lengend Snippet: Distribution of flotillin-2 in C2C12 mouse muscle cell line. C2C12 cells were grown as described in Materials and Methods section. Cells were fixed with methanol and stained with an anti-flotillin-2 antibody ( green , A and C ) and the nuclear dye DAPI ( blue , B and C ). A merged image is shown in C . Note that flotillin-2 is present in C2C12 cells in vesicle-like structures (arrows in A and C ). Scale bar in C represents 20 µm.

    Article Snippet: The sections were blocked in 50 mM NH4 Cl and 3% bovine serum albumin in PBS, pH 8.0 for 30 min, and incubated with a monoclonal anti-flotillin-2 antibody (BD Transduction Laboratories, USA) diluted at 1∶5, followed by a goat anti-mouse 10-η m gold-conjugated (BB International, USA) diluted at 1∶100.

    Techniques: Staining

    Flotillin-2 distribution in multinucleated myotubes. Myogenic cells were grown for 72-flotillin-2 antibodies (green, A and C ) and the nuclear dye DAPI (blue, B and D ). Note that with methanol fixation it is possible to see that flotillin-2 is present in elongated dots in myotubes (arrows in A and C ). Scale bar in D represents 20 µm.

    Journal: PLoS ONE

    Article Title: Differences in the Expression and Distribution of Flotillin-2 in Chick, Mice and Human Muscle Cells

    doi: 10.1371/journal.pone.0103990

    Figure Lengend Snippet: Flotillin-2 distribution in multinucleated myotubes. Myogenic cells were grown for 72-flotillin-2 antibodies (green, A and C ) and the nuclear dye DAPI (blue, B and D ). Note that with methanol fixation it is possible to see that flotillin-2 is present in elongated dots in myotubes (arrows in A and C ). Scale bar in D represents 20 µm.

    Article Snippet: The sections were blocked in 50 mM NH4 Cl and 3% bovine serum albumin in PBS, pH 8.0 for 30 min, and incubated with a monoclonal anti-flotillin-2 antibody (BD Transduction Laboratories, USA) diluted at 1∶5, followed by a goat anti-mouse 10-η m gold-conjugated (BB International, USA) diluted at 1∶100.

    Techniques:

    Tat palmitoylation is performed by DHHC-20. a HEK 293 T cells were cotransfected (1/5) with the indicated myc-tagged human DHHC and Tat. Tat palmitoylation was then assessed using the acyl-RAC technique, UC unbound control, BC bound control, UH unbound hydroxylamine, BH bound hydroxylamine. Palmitoylated Tat is present in the BH fraction. Palmitoylation was calculated as BH/(BH + UH)-BC/(BC + UC), and flotillin-2 was used as a positive control. The graph shows mean ± SEM of n = 3 independent experiments, *** p

    Journal: Nature Communications

    Article Title: Cyclophilin A enables specific HIV-1 Tat palmitoylation and accumulation in uninfected cells

    doi: 10.1038/s41467-018-04674-y

    Figure Lengend Snippet: Tat palmitoylation is performed by DHHC-20. a HEK 293 T cells were cotransfected (1/5) with the indicated myc-tagged human DHHC and Tat. Tat palmitoylation was then assessed using the acyl-RAC technique, UC unbound control, BC bound control, UH unbound hydroxylamine, BH bound hydroxylamine. Palmitoylated Tat is present in the BH fraction. Palmitoylation was calculated as BH/(BH + UH)-BC/(BC + UC), and flotillin-2 was used as a positive control. The graph shows mean ± SEM of n = 3 independent experiments, *** p

    Article Snippet: Klosen, INCI Strasbourg), anti-DHHC-5 (Sigma HPA014670), anti-DHHC-20 (Sigma SAB 4501054), anti-flotillin-2 (sc-28320) and anti-αtubulin (Sigma T5168) were obtained as indicated.

    Techniques: Positive Control

    Flotillin depletion increases diffusion mobility rates of CFP-HA-DAT

    Journal: Traffic (Copenhagen, Denmark)

    Article Title: Flotillins regulate membrane mobility of the dopamine transporter but are not required for its protein kinase C dependent endocytosis

    doi: 10.1111/tra.12059

    Figure Lengend Snippet: Flotillin depletion increases diffusion mobility rates of CFP-HA-DAT

    Article Snippet: Protein G-Sepharose was purchased from Invitrogen. siRNAs to Flotillin-1 and Non-Targeting siRNA were purchased from Qiagen (Valencia, CA); siRNAs to CHC and μ2/AP-2 ( ) and Smart Pools of duplexes to flotillin-1 and flotillin-2 were purchased from Thermo Fisher Scientific.

    Techniques: Diffusion-based Assay

    Co-immunoprecipitation of DAT with flotillin-1 and Eps15

    Journal: Traffic (Copenhagen, Denmark)

    Article Title: Flotillins regulate membrane mobility of the dopamine transporter but are not required for its protein kinase C dependent endocytosis

    doi: 10.1111/tra.12059

    Figure Lengend Snippet: Co-immunoprecipitation of DAT with flotillin-1 and Eps15

    Article Snippet: Protein G-Sepharose was purchased from Invitrogen. siRNAs to Flotillin-1 and Non-Targeting siRNA were purchased from Qiagen (Valencia, CA); siRNAs to CHC and μ2/AP-2 ( ) and Smart Pools of duplexes to flotillin-1 and flotillin-2 were purchased from Thermo Fisher Scientific.

    Techniques: Immunoprecipitation

    PMA-induced down-regulation of DAT in HEK293 cells is blocked by clathrin but not affected by flotillin knockdowns

    Journal: Traffic (Copenhagen, Denmark)

    Article Title: Flotillins regulate membrane mobility of the dopamine transporter but are not required for its protein kinase C dependent endocytosis

    doi: 10.1111/tra.12059

    Figure Lengend Snippet: PMA-induced down-regulation of DAT in HEK293 cells is blocked by clathrin but not affected by flotillin knockdowns

    Article Snippet: Protein G-Sepharose was purchased from Invitrogen. siRNAs to Flotillin-1 and Non-Targeting siRNA were purchased from Qiagen (Valencia, CA); siRNAs to CHC and μ2/AP-2 ( ) and Smart Pools of duplexes to flotillin-1 and flotillin-2 were purchased from Thermo Fisher Scientific.

    Techniques:

    Comparison of localization of flotillin-1 and DAT in dopaminergic neurons

    Journal: Traffic (Copenhagen, Denmark)

    Article Title: Flotillins regulate membrane mobility of the dopamine transporter but are not required for its protein kinase C dependent endocytosis

    doi: 10.1111/tra.12059

    Figure Lengend Snippet: Comparison of localization of flotillin-1 and DAT in dopaminergic neurons

    Article Snippet: Protein G-Sepharose was purchased from Invitrogen. siRNAs to Flotillin-1 and Non-Targeting siRNA were purchased from Qiagen (Valencia, CA); siRNAs to CHC and μ2/AP-2 ( ) and Smart Pools of duplexes to flotillin-1 and flotillin-2 were purchased from Thermo Fisher Scientific.

    Techniques:

    Depletion of both flotillin-1 and flotillin-2 does not inhibit PMA-dependent endocytosis of DAT in HEK293 cells

    Journal: Traffic (Copenhagen, Denmark)

    Article Title: Flotillins regulate membrane mobility of the dopamine transporter but are not required for its protein kinase C dependent endocytosis

    doi: 10.1111/tra.12059

    Figure Lengend Snippet: Depletion of both flotillin-1 and flotillin-2 does not inhibit PMA-dependent endocytosis of DAT in HEK293 cells

    Article Snippet: Protein G-Sepharose was purchased from Invitrogen. siRNAs to Flotillin-1 and Non-Targeting siRNA were purchased from Qiagen (Valencia, CA); siRNAs to CHC and μ2/AP-2 ( ) and Smart Pools of duplexes to flotillin-1 and flotillin-2 were purchased from Thermo Fisher Scientific.

    Techniques:

    Depletion of flotillin-1 and flotillin-2 does not inhibit PMA-dependent endocytosis of DAT in HEK/DAT cells

    Journal: Traffic (Copenhagen, Denmark)

    Article Title: Flotillins regulate membrane mobility of the dopamine transporter but are not required for its protein kinase C dependent endocytosis

    doi: 10.1111/tra.12059

    Figure Lengend Snippet: Depletion of flotillin-1 and flotillin-2 does not inhibit PMA-dependent endocytosis of DAT in HEK/DAT cells

    Article Snippet: Protein G-Sepharose was purchased from Invitrogen. siRNAs to Flotillin-1 and Non-Targeting siRNA were purchased from Qiagen (Valencia, CA); siRNAs to CHC and μ2/AP-2 ( ) and Smart Pools of duplexes to flotillin-1 and flotillin-2 were purchased from Thermo Fisher Scientific.

    Techniques:

    Flotillin-1 siRNA does not inhibit PMA-dependent endocytosis of DAT in HeLa cells

    Journal: Traffic (Copenhagen, Denmark)

    Article Title: Flotillins regulate membrane mobility of the dopamine transporter but are not required for its protein kinase C dependent endocytosis

    doi: 10.1111/tra.12059

    Figure Lengend Snippet: Flotillin-1 siRNA does not inhibit PMA-dependent endocytosis of DAT in HeLa cells

    Article Snippet: Protein G-Sepharose was purchased from Invitrogen. siRNAs to Flotillin-1 and Non-Targeting siRNA were purchased from Qiagen (Valencia, CA); siRNAs to CHC and μ2/AP-2 ( ) and Smart Pools of duplexes to flotillin-1 and flotillin-2 were purchased from Thermo Fisher Scientific.

    Techniques:

    Comparison of localization of flotillin-1 and DAT in HEK293 cells

    Journal: Traffic (Copenhagen, Denmark)

    Article Title: Flotillins regulate membrane mobility of the dopamine transporter but are not required for its protein kinase C dependent endocytosis

    doi: 10.1111/tra.12059

    Figure Lengend Snippet: Comparison of localization of flotillin-1 and DAT in HEK293 cells

    Article Snippet: Protein G-Sepharose was purchased from Invitrogen. siRNAs to Flotillin-1 and Non-Targeting siRNA were purchased from Qiagen (Valencia, CA); siRNAs to CHC and μ2/AP-2 ( ) and Smart Pools of duplexes to flotillin-1 and flotillin-2 were purchased from Thermo Fisher Scientific.

    Techniques:

    Flotillin-1 siRNA does not inhibit PMA-dependent endocytosis of DAT in HEK293 cells

    Journal: Traffic (Copenhagen, Denmark)

    Article Title: Flotillins regulate membrane mobility of the dopamine transporter but are not required for its protein kinase C dependent endocytosis

    doi: 10.1111/tra.12059

    Figure Lengend Snippet: Flotillin-1 siRNA does not inhibit PMA-dependent endocytosis of DAT in HEK293 cells

    Article Snippet: Protein G-Sepharose was purchased from Invitrogen. siRNAs to Flotillin-1 and Non-Targeting siRNA were purchased from Qiagen (Valencia, CA); siRNAs to CHC and μ2/AP-2 ( ) and Smart Pools of duplexes to flotillin-1 and flotillin-2 were purchased from Thermo Fisher Scientific.

    Techniques: