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rabbit polyclonal anti cd63  (Abcam)

 
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    Abcam rabbit polyclonal anti cd63
    KIBRA Knockout reduces secretion of EVs harboring APP-CTFβ/Aβ both in vivo and in vitro . ( a ) WB analysis of small EVs from cell culture supernatants from equal number of KIBRA-KD and CTRL HT22 cells overexpressing APP swe . Whole cell lysates (WCLs) and small EVs were blotted for the EVs markers Alix, <t>CD63,</t> CD9, and APP-CTFβ and for the endoplasmic reticulum marker Calnexin. ( b ) Quantification of EVs protein levels in the small EVs obtained from KIBRA-KD and CTRL cells overexpressing APP swe in three independent experiments. ( c ) Representative NTA traces of EVs isolated from cell culture supernatants from equal numbers of KIBRA-KD and CTRL cells overexpressing APP swe . Quantification of NTA of four independent experiments. ( d ) Simoa analysis was used to measure human Aβ40 and Aβ42 levels in small EVs purified from sucrose step gradient centrifugation from equal numbers of KIBRA-KD and CTRL cells with APP swe overexpression. ( n = 3, from three independent experiments). ( e ) Quantification of exosomal APP-CTFβ levels in the small EVs obtained from KIBRA-KD and CTRL cells overexpressing APP swe in three independent experiments. ( f ) Sucrose step gradient fraction d isolated from the extracellular space of the brain from 3–5-month-old of the indicated groups mice were blotted for the exosomal markers <t>CD63,</t> CD9, Calnexin, and APP-CTFβ. ( g ) Quantification of exosomal protein levels in the small EVs obtained from the indicated groups mice in three independent experiments. ( h ) Human Aβ40 and Aβ42 levels were measured by MSD analysis from sucrose step gradient fraction d isolated from the extracellular space of the brain from 3–5-month-old indicated groups mice ( n = 6, from three biological replicates). ( i ) Quantification of exosomal APP-CTFβ levels in the small EVs obtained from the indicated groups mice in three independent experiments. Data are represented as the mean ± SE. In a - e , ⁎⁎ P < 0.01, ⁎⁎⁎ P < 0.001, and ⁎⁎⁎⁎ P < 0.0001 in two-tailed Student's t test. In f-h , * P < 0.05, ⁎⁎ P <0.01, ⁎⁎⁎ P < 0.001, and ⁎⁎⁎⁎ P < 0.0001 as determined by one-way ANOVA followed by Tukey's post hoc comparisons tests.
    Rabbit Polyclonal Anti Cd63, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 305 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit polyclonal anti cd63/product/Abcam
    Average 94 stars, based on 305 article reviews
    rabbit polyclonal anti cd63 - by Bioz Stars, 2026-03
    94/100 stars

    Images

    1) Product Images from "KIBRA regulates amyloid β metabolism by controlling extracellular vesicles secretion"

    Article Title: KIBRA regulates amyloid β metabolism by controlling extracellular vesicles secretion

    Journal: EBioMedicine

    doi: 10.1016/j.ebiom.2022.103980

    KIBRA Knockout reduces secretion of EVs harboring APP-CTFβ/Aβ both in vivo and in vitro . ( a ) WB analysis of small EVs from cell culture supernatants from equal number of KIBRA-KD and CTRL HT22 cells overexpressing APP swe . Whole cell lysates (WCLs) and small EVs were blotted for the EVs markers Alix, CD63, CD9, and APP-CTFβ and for the endoplasmic reticulum marker Calnexin. ( b ) Quantification of EVs protein levels in the small EVs obtained from KIBRA-KD and CTRL cells overexpressing APP swe in three independent experiments. ( c ) Representative NTA traces of EVs isolated from cell culture supernatants from equal numbers of KIBRA-KD and CTRL cells overexpressing APP swe . Quantification of NTA of four independent experiments. ( d ) Simoa analysis was used to measure human Aβ40 and Aβ42 levels in small EVs purified from sucrose step gradient centrifugation from equal numbers of KIBRA-KD and CTRL cells with APP swe overexpression. ( n = 3, from three independent experiments). ( e ) Quantification of exosomal APP-CTFβ levels in the small EVs obtained from KIBRA-KD and CTRL cells overexpressing APP swe in three independent experiments. ( f ) Sucrose step gradient fraction d isolated from the extracellular space of the brain from 3–5-month-old of the indicated groups mice were blotted for the exosomal markers CD63, CD9, Calnexin, and APP-CTFβ. ( g ) Quantification of exosomal protein levels in the small EVs obtained from the indicated groups mice in three independent experiments. ( h ) Human Aβ40 and Aβ42 levels were measured by MSD analysis from sucrose step gradient fraction d isolated from the extracellular space of the brain from 3–5-month-old indicated groups mice ( n = 6, from three biological replicates). ( i ) Quantification of exosomal APP-CTFβ levels in the small EVs obtained from the indicated groups mice in three independent experiments. Data are represented as the mean ± SE. In a - e , ⁎⁎ P < 0.01, ⁎⁎⁎ P < 0.001, and ⁎⁎⁎⁎ P < 0.0001 in two-tailed Student's t test. In f-h , * P < 0.05, ⁎⁎ P <0.01, ⁎⁎⁎ P < 0.001, and ⁎⁎⁎⁎ P < 0.0001 as determined by one-way ANOVA followed by Tukey's post hoc comparisons tests.
    Figure Legend Snippet: KIBRA Knockout reduces secretion of EVs harboring APP-CTFβ/Aβ both in vivo and in vitro . ( a ) WB analysis of small EVs from cell culture supernatants from equal number of KIBRA-KD and CTRL HT22 cells overexpressing APP swe . Whole cell lysates (WCLs) and small EVs were blotted for the EVs markers Alix, CD63, CD9, and APP-CTFβ and for the endoplasmic reticulum marker Calnexin. ( b ) Quantification of EVs protein levels in the small EVs obtained from KIBRA-KD and CTRL cells overexpressing APP swe in three independent experiments. ( c ) Representative NTA traces of EVs isolated from cell culture supernatants from equal numbers of KIBRA-KD and CTRL cells overexpressing APP swe . Quantification of NTA of four independent experiments. ( d ) Simoa analysis was used to measure human Aβ40 and Aβ42 levels in small EVs purified from sucrose step gradient centrifugation from equal numbers of KIBRA-KD and CTRL cells with APP swe overexpression. ( n = 3, from three independent experiments). ( e ) Quantification of exosomal APP-CTFβ levels in the small EVs obtained from KIBRA-KD and CTRL cells overexpressing APP swe in three independent experiments. ( f ) Sucrose step gradient fraction d isolated from the extracellular space of the brain from 3–5-month-old of the indicated groups mice were blotted for the exosomal markers CD63, CD9, Calnexin, and APP-CTFβ. ( g ) Quantification of exosomal protein levels in the small EVs obtained from the indicated groups mice in three independent experiments. ( h ) Human Aβ40 and Aβ42 levels were measured by MSD analysis from sucrose step gradient fraction d isolated from the extracellular space of the brain from 3–5-month-old indicated groups mice ( n = 6, from three biological replicates). ( i ) Quantification of exosomal APP-CTFβ levels in the small EVs obtained from the indicated groups mice in three independent experiments. Data are represented as the mean ± SE. In a - e , ⁎⁎ P < 0.01, ⁎⁎⁎ P < 0.001, and ⁎⁎⁎⁎ P < 0.0001 in two-tailed Student's t test. In f-h , * P < 0.05, ⁎⁎ P <0.01, ⁎⁎⁎ P < 0.001, and ⁎⁎⁎⁎ P < 0.0001 as determined by one-way ANOVA followed by Tukey's post hoc comparisons tests.

    Techniques Used: Knock-Out, In Vivo, In Vitro, Cell Culture, Marker, Isolation, Purification, Gradient Centrifugation, Over Expression, Two Tailed Test

    KIBRA knockout causes increases in number of MVBs harboring AβPP/Aβ. ( a and b ) Representative electron microscopic images of the hippocampus ( a ) and cortex ( b ) area in KIBRA −/− 5XFAD and 5XFAD mice. Scale bar = 500 nm. Red arrows indicate MVBs containing typical ILVs. Quantification analysis of the number of MVBs per cell profile and the number of ILVs per MVB. ILVs and MVBs in 28-31 profiles of different cells were counted in a blind manner and only MVBs containing typical ILVs were counted. ( c ) Confocal microscopy analysis of Aβ/AβPP (anti-Aβ, clone 4G8, red) with CD63 (green) in brain slices of KIBRA −/− 5XFAD and 5XFAD mice. Scale bar = 10 µm. Right panel represents overlap coefficient per cell. Data are represented as the mean ± SE. * P < 0.05, ⁎⁎⁎ P < 0.001, and ⁎⁎⁎⁎ P < 0.0001 in two-tailed Student's t test.
    Figure Legend Snippet: KIBRA knockout causes increases in number of MVBs harboring AβPP/Aβ. ( a and b ) Representative electron microscopic images of the hippocampus ( a ) and cortex ( b ) area in KIBRA −/− 5XFAD and 5XFAD mice. Scale bar = 500 nm. Red arrows indicate MVBs containing typical ILVs. Quantification analysis of the number of MVBs per cell profile and the number of ILVs per MVB. ILVs and MVBs in 28-31 profiles of different cells were counted in a blind manner and only MVBs containing typical ILVs were counted. ( c ) Confocal microscopy analysis of Aβ/AβPP (anti-Aβ, clone 4G8, red) with CD63 (green) in brain slices of KIBRA −/− 5XFAD and 5XFAD mice. Scale bar = 10 µm. Right panel represents overlap coefficient per cell. Data are represented as the mean ± SE. * P < 0.05, ⁎⁎⁎ P < 0.001, and ⁎⁎⁎⁎ P < 0.0001 in two-tailed Student's t test.

    Techniques Used: Knock-Out, Confocal Microscopy, Two Tailed Test

    KIBRA Knockout leads to AβPP/Aβ aggregation and degradation by lysosomes. ( a and b ) Confocal microscopy analysis of MVBs marker CD63 (green) and lysosomal marker LAMP2 (red) co-localization in cortical layers 5 ( a ) and hippocampal CA 1 - 3 ( b ) regions of WT, KIBRA −/− , 5XFAD, and KIBRA −/− 5XFAD mice. Nuclei were stained with DAPI. Scale bar in left panel = 20 µm. Scale bar in right panel = 10 µm. ( c ) Quantification analysis of overlap coefficient per view ( n = 7 in the cortex and n = 5 in the hippocampus). ( d ) WB analysis of lysosomal marker Lamp2 and Cathepsin D levels in brain tissue lysates from cortical and hippocampal regions of 3–5-month-old mice. ( e ) Quantification analysis of lysosomal membrane protein levels (Lamp2, left panel) and ratio of CatD/proCatD (right panel) in cortical and hippocampal regions from 3–5-month-old mice of the indicated genotypes. Quantification results were plotted as dot plots, showing the mean ± SE of at least three independent experiments. * P < 0.05, ⁎⁎ P < 0.01, ⁎⁎⁎⁎ P < 0.0001 as determined by a two-tailed t test. ( f ) Confocal microscopy analysis of lysosomal membrane marker Lamp2 (red) with Aβ/AβPP (anti-Aβ, clone 4G8, green) in brain slice of 5XFAD and KIBRA −/− 5XFAD mice. Scale bar = 10 µm. The overlap coefficient per cell was quantified ( n = 15). ( g ) Confocal microscopy analysis of Cathepsin D (red) with Aβ/AβPP (anti-Aβ, clone 4G8, green) in brain slice of 5XFAD and KIBRA −/− 5XFAD mice. Scale bar = 10 µm. Overlap coefficient per cell was quantified ( n = 15). f-g . Quantification result were plotted as dot plots, showing the mean ± SE. ⁎⁎⁎ P < 0.001, ⁎⁎⁎⁎ P < 0.0001 as determined by a two-tailed student's t test.
    Figure Legend Snippet: KIBRA Knockout leads to AβPP/Aβ aggregation and degradation by lysosomes. ( a and b ) Confocal microscopy analysis of MVBs marker CD63 (green) and lysosomal marker LAMP2 (red) co-localization in cortical layers 5 ( a ) and hippocampal CA 1 - 3 ( b ) regions of WT, KIBRA −/− , 5XFAD, and KIBRA −/− 5XFAD mice. Nuclei were stained with DAPI. Scale bar in left panel = 20 µm. Scale bar in right panel = 10 µm. ( c ) Quantification analysis of overlap coefficient per view ( n = 7 in the cortex and n = 5 in the hippocampus). ( d ) WB analysis of lysosomal marker Lamp2 and Cathepsin D levels in brain tissue lysates from cortical and hippocampal regions of 3–5-month-old mice. ( e ) Quantification analysis of lysosomal membrane protein levels (Lamp2, left panel) and ratio of CatD/proCatD (right panel) in cortical and hippocampal regions from 3–5-month-old mice of the indicated genotypes. Quantification results were plotted as dot plots, showing the mean ± SE of at least three independent experiments. * P < 0.05, ⁎⁎ P < 0.01, ⁎⁎⁎⁎ P < 0.0001 as determined by a two-tailed t test. ( f ) Confocal microscopy analysis of lysosomal membrane marker Lamp2 (red) with Aβ/AβPP (anti-Aβ, clone 4G8, green) in brain slice of 5XFAD and KIBRA −/− 5XFAD mice. Scale bar = 10 µm. The overlap coefficient per cell was quantified ( n = 15). ( g ) Confocal microscopy analysis of Cathepsin D (red) with Aβ/AβPP (anti-Aβ, clone 4G8, green) in brain slice of 5XFAD and KIBRA −/− 5XFAD mice. Scale bar = 10 µm. Overlap coefficient per cell was quantified ( n = 15). f-g . Quantification result were plotted as dot plots, showing the mean ± SE. ⁎⁎⁎ P < 0.001, ⁎⁎⁎⁎ P < 0.0001 as determined by a two-tailed student's t test.

    Techniques Used: Knock-Out, Confocal Microscopy, Marker, Staining, Two Tailed Test, Slice Preparation

    Inhibition of lysosome function in KIBRA knockout cells rescues secretion of EVs harboring APP-CTFβ/Aβ. ( a ) WB analysis of WCLs in KIBRA-KD and CTRL cells overexpressing APP swe , and treated with 20 nM bafilomycinA1 (BafA1) for 12 h, when indicated, and WCLs were blotted for Alix, CD63, and CD9. ( b ) Representative NTA traces of EVs derived from KIBRA-KD and CTRL cells overexpressing APP swe , and treated with BafA1. Right panel represents quantification of NTA of three independent experiments. ( c ) Human Aβ40 and Aβ42 levels measured by Simoa analysis from small EVs purified from sucrose step gradient centrifugation from equal number of KIBRA-KD and CTRL cells overexpressing APP swe, and treated with BafA1. ( n = 3, from three independent experiments). ( d ) WB analysis of EVs secretion in KIBRA-KD and CTRL cells overexpressing APP swe , and treated with 20 nM bafilomycinA1 (BafA1) for 12 h, when indicated. EVs were blotted for Alix, CD63, CD9, and APP-CTFβ. ( e ) Quantification of EVs protein levels in the small EVs obtained from KIBRA-KD and CTRL cells overexpressing APP swe , and treated with lysosome inhibitor BafA1 in three independent experiments. ( f ) Quantification of APP-CTFβ levels in the small EVs obtained from KIBRA-KD and CTRL cells overexpressing APP swe , and treated with lysosome inhibitor BafA1 in three independent experiments. ( g and h ) WB analysis of EVs secretion in KIBRA-KD and CTRL cells overexpressing APP swe transfected with control small interfering RNAs (siRNAs) or siRNAs targeting Rab7. WCLs ( g ) and EVs ( h ) were blotted for Alix, CD63, CD9, Rab7, and APP-CTFβ. ( i ) Quantification of EVs protein levels (Alix, CD63, and CD9) in the small EVs obtained from KIBRA-KD and CTRL cells overexpressing APP swe , and transfected with siRNAs in four independent experiments. ( j ) Quantification of APP-CTFβ levels in the small EVs obtained from KIBRA-KD and CTRL cells overexpressing APP swe , and transfected with siRNAs in three independent experiments. ( k ) Representative NTA traces of EVs derived from KIBRA-KD and CTRL cells overexpressing APP swe , and treated with siRNAs targeting Rab7. Right panel represents quantification of NTA of three independent experiments. ( l ) Human Aβ40 and Aβ42 levels measured by Simoa analysis from small EVs purified from sucrose step gradient centrifugation from equal numbers of KIBRA-KD and CTRL cells overexpressing APP swe , and treated with siRNAs in three independent experiments. The quantification results were plotted as dot plots, showing the mean ± SE. * P < 0.05, ⁎⁎ P < 0.01, ⁎⁎⁎ P < 0.001, ⁎⁎⁎⁎ P < 0.0001, n.s., not significant ( P > 0.05) as determined by one-way ANOVA followed by Tukey's post hoc comparisons tests.
    Figure Legend Snippet: Inhibition of lysosome function in KIBRA knockout cells rescues secretion of EVs harboring APP-CTFβ/Aβ. ( a ) WB analysis of WCLs in KIBRA-KD and CTRL cells overexpressing APP swe , and treated with 20 nM bafilomycinA1 (BafA1) for 12 h, when indicated, and WCLs were blotted for Alix, CD63, and CD9. ( b ) Representative NTA traces of EVs derived from KIBRA-KD and CTRL cells overexpressing APP swe , and treated with BafA1. Right panel represents quantification of NTA of three independent experiments. ( c ) Human Aβ40 and Aβ42 levels measured by Simoa analysis from small EVs purified from sucrose step gradient centrifugation from equal number of KIBRA-KD and CTRL cells overexpressing APP swe, and treated with BafA1. ( n = 3, from three independent experiments). ( d ) WB analysis of EVs secretion in KIBRA-KD and CTRL cells overexpressing APP swe , and treated with 20 nM bafilomycinA1 (BafA1) for 12 h, when indicated. EVs were blotted for Alix, CD63, CD9, and APP-CTFβ. ( e ) Quantification of EVs protein levels in the small EVs obtained from KIBRA-KD and CTRL cells overexpressing APP swe , and treated with lysosome inhibitor BafA1 in three independent experiments. ( f ) Quantification of APP-CTFβ levels in the small EVs obtained from KIBRA-KD and CTRL cells overexpressing APP swe , and treated with lysosome inhibitor BafA1 in three independent experiments. ( g and h ) WB analysis of EVs secretion in KIBRA-KD and CTRL cells overexpressing APP swe transfected with control small interfering RNAs (siRNAs) or siRNAs targeting Rab7. WCLs ( g ) and EVs ( h ) were blotted for Alix, CD63, CD9, Rab7, and APP-CTFβ. ( i ) Quantification of EVs protein levels (Alix, CD63, and CD9) in the small EVs obtained from KIBRA-KD and CTRL cells overexpressing APP swe , and transfected with siRNAs in four independent experiments. ( j ) Quantification of APP-CTFβ levels in the small EVs obtained from KIBRA-KD and CTRL cells overexpressing APP swe , and transfected with siRNAs in three independent experiments. ( k ) Representative NTA traces of EVs derived from KIBRA-KD and CTRL cells overexpressing APP swe , and treated with siRNAs targeting Rab7. Right panel represents quantification of NTA of three independent experiments. ( l ) Human Aβ40 and Aβ42 levels measured by Simoa analysis from small EVs purified from sucrose step gradient centrifugation from equal numbers of KIBRA-KD and CTRL cells overexpressing APP swe , and treated with siRNAs in three independent experiments. The quantification results were plotted as dot plots, showing the mean ± SE. * P < 0.05, ⁎⁎ P < 0.01, ⁎⁎⁎ P < 0.001, ⁎⁎⁎⁎ P < 0.0001, n.s., not significant ( P > 0.05) as determined by one-way ANOVA followed by Tukey's post hoc comparisons tests.

    Techniques Used: Inhibition, Knock-Out, Derivative Assay, Purification, Gradient Centrifugation, Transfection



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    94
    Abcam rabbit polyclonal antibodies to cd63
    KIBRA Knockout reduces secretion of EVs harboring APP-CTFβ/Aβ both in vivo and in vitro . ( a ) WB analysis of small EVs from cell culture supernatants from equal number of KIBRA-KD and CTRL HT22 cells overexpressing APP swe . Whole cell lysates (WCLs) and small EVs were blotted for the EVs markers Alix, <t>CD63,</t> CD9, and APP-CTFβ and for the endoplasmic reticulum marker Calnexin. ( b ) Quantification of EVs protein levels in the small EVs obtained from KIBRA-KD and CTRL cells overexpressing APP swe in three independent experiments. ( c ) Representative NTA traces of EVs isolated from cell culture supernatants from equal numbers of KIBRA-KD and CTRL cells overexpressing APP swe . Quantification of NTA of four independent experiments. ( d ) Simoa analysis was used to measure human Aβ40 and Aβ42 levels in small EVs purified from sucrose step gradient centrifugation from equal numbers of KIBRA-KD and CTRL cells with APP swe overexpression. ( n = 3, from three independent experiments). ( e ) Quantification of exosomal APP-CTFβ levels in the small EVs obtained from KIBRA-KD and CTRL cells overexpressing APP swe in three independent experiments. ( f ) Sucrose step gradient fraction d isolated from the extracellular space of the brain from 3–5-month-old of the indicated groups mice were blotted for the exosomal markers <t>CD63,</t> CD9, Calnexin, and APP-CTFβ. ( g ) Quantification of exosomal protein levels in the small EVs obtained from the indicated groups mice in three independent experiments. ( h ) Human Aβ40 and Aβ42 levels were measured by MSD analysis from sucrose step gradient fraction d isolated from the extracellular space of the brain from 3–5-month-old indicated groups mice ( n = 6, from three biological replicates). ( i ) Quantification of exosomal APP-CTFβ levels in the small EVs obtained from the indicated groups mice in three independent experiments. Data are represented as the mean ± SE. In a - e , ⁎⁎ P < 0.01, ⁎⁎⁎ P < 0.001, and ⁎⁎⁎⁎ P < 0.0001 in two-tailed Student's t test. In f-h , * P < 0.05, ⁎⁎ P <0.01, ⁎⁎⁎ P < 0.001, and ⁎⁎⁎⁎ P < 0.0001 as determined by one-way ANOVA followed by Tukey's post hoc comparisons tests.
    Rabbit Polyclonal Antibodies To Cd63, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    rabbit polyclonal antibodies to cd63 - by Bioz Stars, 2026-03
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    human cd63 antibody  (Cusabio)
    93
    Cusabio human cd63 antibody
    Primary antibodies used for Western blotting.
    Human Cd63 Antibody, supplied by Cusabio, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    rabbit anti-human cd63 polyclonal antibody  (Proteintech)
    90
    Proteintech rabbit anti-human cd63 polyclonal antibody
    Primary antibodies used for Western blotting.
    Rabbit Anti Human Cd63 Polyclonal Antibody, supplied by Proteintech, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Western blotting assay for the verification of the BMSC-MVs. CD63, CD73, and CD81 with approximately 50, 75, and 26 kDa protein bands, respectively, were detected

    Journal: Journal of Cancer Research and Clinical Oncology

    Article Title: Effect of human bone marrow mesenchymal stem cell-derived microvesicles on the apoptosis of the multiple myeloma cell line U266

    doi: 10.1007/s00432-024-05822-2

    Figure Lengend Snippet: Western blotting assay for the verification of the BMSC-MVs. CD63, CD73, and CD81 with approximately 50, 75, and 26 kDa protein bands, respectively, were detected

    Article Snippet: Next, the membranes were separately incubated with the following primary antibodies: rabbit polyclonal anti-human CD63 antibodies (cat. No. 11,271-T16; Sino Biological, China), rabbit monoclonal anti-human CD73 antibodies (cat No. ab124725; Abcam, MA, United States), and rabbit polyclonal anti-human CD81 antibodies (cat No. ab155760; Abcam, MA, United States) as per the suppliers’ instructions.

    Techniques: Western Blot

    Fig. 3. CHKA mediates exosomes secretion in glioma cells. A-C Western blot (WB) analysis of exosome marker proteins CD63 and TSG101 in exosomes isolated from the supernatant of glioma cells in shCHKA and shNC group along with the negative control protein Calnexin. D Exosome protein concentration in the shCHKA and shNC groups of glioma cells using the BCA assay. E Electron microscopy was performed on shCHKA and shNC cells in glioma cells (Scale bars = 1 μm). F The number of multivesicular bodies (MVBs) per glioma cell profile. G The number of intraluminal vesicles (ILVs) per MVB profile in glioma cells. *P < 0.05, **P < 0.01, ***P < 0.001. shCHKA: CHKA knockdown group, shNC: con trol group.

    Journal: Biochemical and biophysical research communications

    Article Title: Choline kinase alpha regulates autophagy-associated exosome release to promote glioma cell progression.

    doi: 10.1016/j.bbrc.2024.151269

    Figure Lengend Snippet: Fig. 3. CHKA mediates exosomes secretion in glioma cells. A-C Western blot (WB) analysis of exosome marker proteins CD63 and TSG101 in exosomes isolated from the supernatant of glioma cells in shCHKA and shNC group along with the negative control protein Calnexin. D Exosome protein concentration in the shCHKA and shNC groups of glioma cells using the BCA assay. E Electron microscopy was performed on shCHKA and shNC cells in glioma cells (Scale bars = 1 μm). F The number of multivesicular bodies (MVBs) per glioma cell profile. G The number of intraluminal vesicles (ILVs) per MVB profile in glioma cells. *P < 0.05, **P < 0.01, ***P < 0.001. shCHKA: CHKA knockdown group, shNC: con trol group.

    Article Snippet: Antibodies used in the Western blotting process included: rabbit antihuman CHKA polyclonal antibody (abcam, USA), rabbit anti-human GAPDH polyclonal antibody (Affinity, China), rabbit anti-human CD63 polyclonal antibody (Proteintech, China), rabbit anti-human TSG101 polyclonal antibody(Proteintech, China), rabbit anti-human calnexin polyclonal antibody(Proteintech, China), rabbit anti-human LC3B polyclonal antibody(Sigma, USA), and rabbit anti-human P62/SQSTM1 polyclonal antibody(Sigma, USA).

    Techniques: Western Blot, Marker, Isolation, Negative Control, Protein Concentration, BIA-KA, Electron Microscopy, Knockdown

    Fig. 4. CHKA knockdown Inhibited Proliferation, Migration and invasion of glioma Cells by Blocking Exosome Secretion. A Western blot (WB) analysis of exosome marker proteins CD63 and TSG101 in exosomes isolated from the supernatant of glioma cells in DMSO and GW4869 group. (BC) The proliferation of glioma cells U87MG and U251 in DMSO and GW4869 group, the migration ability of glioma cells U87MG (DE) and U251 (DF) in DMSO and GW4869 group (Scale bars = 200 μm). GH The invasion ability of glioma cells U87MG and U251 in DMSO and GW4869 group(Scale bars = 100 μm). *P < 0.05, **P < 0.01, ***P < 0.001.

    Journal: Biochemical and biophysical research communications

    Article Title: Choline kinase alpha regulates autophagy-associated exosome release to promote glioma cell progression.

    doi: 10.1016/j.bbrc.2024.151269

    Figure Lengend Snippet: Fig. 4. CHKA knockdown Inhibited Proliferation, Migration and invasion of glioma Cells by Blocking Exosome Secretion. A Western blot (WB) analysis of exosome marker proteins CD63 and TSG101 in exosomes isolated from the supernatant of glioma cells in DMSO and GW4869 group. (BC) The proliferation of glioma cells U87MG and U251 in DMSO and GW4869 group, the migration ability of glioma cells U87MG (DE) and U251 (DF) in DMSO and GW4869 group (Scale bars = 200 μm). GH The invasion ability of glioma cells U87MG and U251 in DMSO and GW4869 group(Scale bars = 100 μm). *P < 0.05, **P < 0.01, ***P < 0.001.

    Article Snippet: Antibodies used in the Western blotting process included: rabbit antihuman CHKA polyclonal antibody (abcam, USA), rabbit anti-human GAPDH polyclonal antibody (Affinity, China), rabbit anti-human CD63 polyclonal antibody (Proteintech, China), rabbit anti-human TSG101 polyclonal antibody(Proteintech, China), rabbit anti-human calnexin polyclonal antibody(Proteintech, China), rabbit anti-human LC3B polyclonal antibody(Sigma, USA), and rabbit anti-human P62/SQSTM1 polyclonal antibody(Sigma, USA).

    Techniques: Knockdown, Migration, Blocking Assay, Western Blot, Marker, Isolation

    Fig. 6. CHKA attenuates exosomes release through Autophagy-Mediated mechanism AB Western blot (WB) analysis of exosome marker proteins CD63 and TSG101 in exosomes isolated from the supernatant of U87MG cells in shNC + DMSO, shCHKA + DMSO and shCHKA + Baf-A1 group. AC Western blot (WB) analysis of exosome marker proteins CD63 and TSG101 in exosomes isolated from the supernatant of U251 cells in shNC + DMSO, shCHKA + DMSO and shCHKA + Baf-A1 group. D Exosome protein concentration in the s shNC + DMSO, shCHKA + DMSO and shCHKA + Baf-A1 groups of glioma cells using the BCA assay. **P < 0.01, ***P < 0.001. Baf-A1: Bafilomycin A1.

    Journal: Biochemical and biophysical research communications

    Article Title: Choline kinase alpha regulates autophagy-associated exosome release to promote glioma cell progression.

    doi: 10.1016/j.bbrc.2024.151269

    Figure Lengend Snippet: Fig. 6. CHKA attenuates exosomes release through Autophagy-Mediated mechanism AB Western blot (WB) analysis of exosome marker proteins CD63 and TSG101 in exosomes isolated from the supernatant of U87MG cells in shNC + DMSO, shCHKA + DMSO and shCHKA + Baf-A1 group. AC Western blot (WB) analysis of exosome marker proteins CD63 and TSG101 in exosomes isolated from the supernatant of U251 cells in shNC + DMSO, shCHKA + DMSO and shCHKA + Baf-A1 group. D Exosome protein concentration in the s shNC + DMSO, shCHKA + DMSO and shCHKA + Baf-A1 groups of glioma cells using the BCA assay. **P < 0.01, ***P < 0.001. Baf-A1: Bafilomycin A1.

    Article Snippet: Antibodies used in the Western blotting process included: rabbit antihuman CHKA polyclonal antibody (abcam, USA), rabbit anti-human GAPDH polyclonal antibody (Affinity, China), rabbit anti-human CD63 polyclonal antibody (Proteintech, China), rabbit anti-human TSG101 polyclonal antibody(Proteintech, China), rabbit anti-human calnexin polyclonal antibody(Proteintech, China), rabbit anti-human LC3B polyclonal antibody(Sigma, USA), and rabbit anti-human P62/SQSTM1 polyclonal antibody(Sigma, USA).

    Techniques: Western Blot, Marker, Isolation, Protein Concentration, BIA-KA

    KIBRA Knockout reduces secretion of EVs harboring APP-CTFβ/Aβ both in vivo and in vitro . ( a ) WB analysis of small EVs from cell culture supernatants from equal number of KIBRA-KD and CTRL HT22 cells overexpressing APP swe . Whole cell lysates (WCLs) and small EVs were blotted for the EVs markers Alix, CD63, CD9, and APP-CTFβ and for the endoplasmic reticulum marker Calnexin. ( b ) Quantification of EVs protein levels in the small EVs obtained from KIBRA-KD and CTRL cells overexpressing APP swe in three independent experiments. ( c ) Representative NTA traces of EVs isolated from cell culture supernatants from equal numbers of KIBRA-KD and CTRL cells overexpressing APP swe . Quantification of NTA of four independent experiments. ( d ) Simoa analysis was used to measure human Aβ40 and Aβ42 levels in small EVs purified from sucrose step gradient centrifugation from equal numbers of KIBRA-KD and CTRL cells with APP swe overexpression. ( n = 3, from three independent experiments). ( e ) Quantification of exosomal APP-CTFβ levels in the small EVs obtained from KIBRA-KD and CTRL cells overexpressing APP swe in three independent experiments. ( f ) Sucrose step gradient fraction d isolated from the extracellular space of the brain from 3–5-month-old of the indicated groups mice were blotted for the exosomal markers CD63, CD9, Calnexin, and APP-CTFβ. ( g ) Quantification of exosomal protein levels in the small EVs obtained from the indicated groups mice in three independent experiments. ( h ) Human Aβ40 and Aβ42 levels were measured by MSD analysis from sucrose step gradient fraction d isolated from the extracellular space of the brain from 3–5-month-old indicated groups mice ( n = 6, from three biological replicates). ( i ) Quantification of exosomal APP-CTFβ levels in the small EVs obtained from the indicated groups mice in three independent experiments. Data are represented as the mean ± SE. In a - e , ⁎⁎ P < 0.01, ⁎⁎⁎ P < 0.001, and ⁎⁎⁎⁎ P < 0.0001 in two-tailed Student's t test. In f-h , * P < 0.05, ⁎⁎ P <0.01, ⁎⁎⁎ P < 0.001, and ⁎⁎⁎⁎ P < 0.0001 as determined by one-way ANOVA followed by Tukey's post hoc comparisons tests.

    Journal: EBioMedicine

    Article Title: KIBRA regulates amyloid β metabolism by controlling extracellular vesicles secretion

    doi: 10.1016/j.ebiom.2022.103980

    Figure Lengend Snippet: KIBRA Knockout reduces secretion of EVs harboring APP-CTFβ/Aβ both in vivo and in vitro . ( a ) WB analysis of small EVs from cell culture supernatants from equal number of KIBRA-KD and CTRL HT22 cells overexpressing APP swe . Whole cell lysates (WCLs) and small EVs were blotted for the EVs markers Alix, CD63, CD9, and APP-CTFβ and for the endoplasmic reticulum marker Calnexin. ( b ) Quantification of EVs protein levels in the small EVs obtained from KIBRA-KD and CTRL cells overexpressing APP swe in three independent experiments. ( c ) Representative NTA traces of EVs isolated from cell culture supernatants from equal numbers of KIBRA-KD and CTRL cells overexpressing APP swe . Quantification of NTA of four independent experiments. ( d ) Simoa analysis was used to measure human Aβ40 and Aβ42 levels in small EVs purified from sucrose step gradient centrifugation from equal numbers of KIBRA-KD and CTRL cells with APP swe overexpression. ( n = 3, from three independent experiments). ( e ) Quantification of exosomal APP-CTFβ levels in the small EVs obtained from KIBRA-KD and CTRL cells overexpressing APP swe in three independent experiments. ( f ) Sucrose step gradient fraction d isolated from the extracellular space of the brain from 3–5-month-old of the indicated groups mice were blotted for the exosomal markers CD63, CD9, Calnexin, and APP-CTFβ. ( g ) Quantification of exosomal protein levels in the small EVs obtained from the indicated groups mice in three independent experiments. ( h ) Human Aβ40 and Aβ42 levels were measured by MSD analysis from sucrose step gradient fraction d isolated from the extracellular space of the brain from 3–5-month-old indicated groups mice ( n = 6, from three biological replicates). ( i ) Quantification of exosomal APP-CTFβ levels in the small EVs obtained from the indicated groups mice in three independent experiments. Data are represented as the mean ± SE. In a - e , ⁎⁎ P < 0.01, ⁎⁎⁎ P < 0.001, and ⁎⁎⁎⁎ P < 0.0001 in two-tailed Student's t test. In f-h , * P < 0.05, ⁎⁎ P <0.01, ⁎⁎⁎ P < 0.001, and ⁎⁎⁎⁎ P < 0.0001 as determined by one-way ANOVA followed by Tukey's post hoc comparisons tests.

    Article Snippet: The primary antibodies were obtained from the following sources: mouse monoclonal anti-KIBRA (clone 2A5, provided by Prof Jixin Dong's lab), as previously described ; mouse monoclonal anti-Alix (Cell Signaling Technology Cat# 2171; Research Resource Identifiers [RRID]: AB_2299455, Danvers, MA, USA; 1:1000 in western blot [WB]); rabbit monoclonal anti-CD63 (Abcam Cat# ab217345; RRID: AB_2754982, Cambridge, UK; 1:1000 in WB and 1:100 in immunofluorescence [IF]); rabbit polyclonal anti-CD63 (Abcam Cat# ab216130, RRID not available; 1:25 in IF); mouse monoclonal anti-APP C1/6.1 C-terminal fragment (CTFs) (Biolegend Cat# 802803, RRID: AB_2715853, San Diego, CA, USA; 1:2000 in WB); rabbit monoclonal anti-BACE1 (Abcam Cat# ab108394, RRID: AB_10861218; 1:1000 in WB); Alexa Fluor 594 anti-4G8 (Biolegend Cat# 800715, RRID: AB_2721291; 1:100 in IF); mouse monoclonal anti-4G8 (Biolegend Cat# 800712, RRID: AB_2734548; 1:400 in IF); mouse monoclonal anti-6E10 (Biolegend Cat# 803004, RRID: AB_2715854; 1:1000 in WB); rabbit polyclonal anti-calnexin (Abcam Cat# ab22595, RRID: AB_2069006; 1:1000 in WB); rabbit monoclonal anti-CD9 (Abcam Cat# ab92726, RRID: AB_10561589; 1:1000 in WB); rabbit monoclonal anti-Cathepsin D (Abcam Cat# ab75852, RRID: AB_1523267; 1:1000 in WB and 1:100 in IF); rabbit monoclonal anti-Rab7 (Abcam Cat# ab137029, RRID: AB_2629474; 1:1000 in WB); rabbit polyclonal anti-oligomer A11 (Thermo Fisher Scientific Cat# AHB0052, RRID: AB_2536236; Waltham, MA, USA; 1:1000 in Dot blots); rat monoclonal anti-LAMP2 (Abcam Cat# ab13524, RRID: AB_2134736; 1:1000 in WB and 1:100 in IF); rabbit monoclonal anti-beta III Tubulin (Abcam Cat# ab18207; RRID: AB_ 444319; 1:300 in IF); rabbit monoclonal anti-NeuN (Abcam Cat# ab177487, RRID: AB_2532109; 1:200 in IF).

    Techniques: Knock-Out, In Vivo, In Vitro, Cell Culture, Marker, Isolation, Purification, Gradient Centrifugation, Over Expression, Two Tailed Test

    KIBRA knockout causes increases in number of MVBs harboring AβPP/Aβ. ( a and b ) Representative electron microscopic images of the hippocampus ( a ) and cortex ( b ) area in KIBRA −/− 5XFAD and 5XFAD mice. Scale bar = 500 nm. Red arrows indicate MVBs containing typical ILVs. Quantification analysis of the number of MVBs per cell profile and the number of ILVs per MVB. ILVs and MVBs in 28-31 profiles of different cells were counted in a blind manner and only MVBs containing typical ILVs were counted. ( c ) Confocal microscopy analysis of Aβ/AβPP (anti-Aβ, clone 4G8, red) with CD63 (green) in brain slices of KIBRA −/− 5XFAD and 5XFAD mice. Scale bar = 10 µm. Right panel represents overlap coefficient per cell. Data are represented as the mean ± SE. * P < 0.05, ⁎⁎⁎ P < 0.001, and ⁎⁎⁎⁎ P < 0.0001 in two-tailed Student's t test.

    Journal: EBioMedicine

    Article Title: KIBRA regulates amyloid β metabolism by controlling extracellular vesicles secretion

    doi: 10.1016/j.ebiom.2022.103980

    Figure Lengend Snippet: KIBRA knockout causes increases in number of MVBs harboring AβPP/Aβ. ( a and b ) Representative electron microscopic images of the hippocampus ( a ) and cortex ( b ) area in KIBRA −/− 5XFAD and 5XFAD mice. Scale bar = 500 nm. Red arrows indicate MVBs containing typical ILVs. Quantification analysis of the number of MVBs per cell profile and the number of ILVs per MVB. ILVs and MVBs in 28-31 profiles of different cells were counted in a blind manner and only MVBs containing typical ILVs were counted. ( c ) Confocal microscopy analysis of Aβ/AβPP (anti-Aβ, clone 4G8, red) with CD63 (green) in brain slices of KIBRA −/− 5XFAD and 5XFAD mice. Scale bar = 10 µm. Right panel represents overlap coefficient per cell. Data are represented as the mean ± SE. * P < 0.05, ⁎⁎⁎ P < 0.001, and ⁎⁎⁎⁎ P < 0.0001 in two-tailed Student's t test.

    Article Snippet: The primary antibodies were obtained from the following sources: mouse monoclonal anti-KIBRA (clone 2A5, provided by Prof Jixin Dong's lab), as previously described ; mouse monoclonal anti-Alix (Cell Signaling Technology Cat# 2171; Research Resource Identifiers [RRID]: AB_2299455, Danvers, MA, USA; 1:1000 in western blot [WB]); rabbit monoclonal anti-CD63 (Abcam Cat# ab217345; RRID: AB_2754982, Cambridge, UK; 1:1000 in WB and 1:100 in immunofluorescence [IF]); rabbit polyclonal anti-CD63 (Abcam Cat# ab216130, RRID not available; 1:25 in IF); mouse monoclonal anti-APP C1/6.1 C-terminal fragment (CTFs) (Biolegend Cat# 802803, RRID: AB_2715853, San Diego, CA, USA; 1:2000 in WB); rabbit monoclonal anti-BACE1 (Abcam Cat# ab108394, RRID: AB_10861218; 1:1000 in WB); Alexa Fluor 594 anti-4G8 (Biolegend Cat# 800715, RRID: AB_2721291; 1:100 in IF); mouse monoclonal anti-4G8 (Biolegend Cat# 800712, RRID: AB_2734548; 1:400 in IF); mouse monoclonal anti-6E10 (Biolegend Cat# 803004, RRID: AB_2715854; 1:1000 in WB); rabbit polyclonal anti-calnexin (Abcam Cat# ab22595, RRID: AB_2069006; 1:1000 in WB); rabbit monoclonal anti-CD9 (Abcam Cat# ab92726, RRID: AB_10561589; 1:1000 in WB); rabbit monoclonal anti-Cathepsin D (Abcam Cat# ab75852, RRID: AB_1523267; 1:1000 in WB and 1:100 in IF); rabbit monoclonal anti-Rab7 (Abcam Cat# ab137029, RRID: AB_2629474; 1:1000 in WB); rabbit polyclonal anti-oligomer A11 (Thermo Fisher Scientific Cat# AHB0052, RRID: AB_2536236; Waltham, MA, USA; 1:1000 in Dot blots); rat monoclonal anti-LAMP2 (Abcam Cat# ab13524, RRID: AB_2134736; 1:1000 in WB and 1:100 in IF); rabbit monoclonal anti-beta III Tubulin (Abcam Cat# ab18207; RRID: AB_ 444319; 1:300 in IF); rabbit monoclonal anti-NeuN (Abcam Cat# ab177487, RRID: AB_2532109; 1:200 in IF).

    Techniques: Knock-Out, Confocal Microscopy, Two Tailed Test

    KIBRA Knockout leads to AβPP/Aβ aggregation and degradation by lysosomes. ( a and b ) Confocal microscopy analysis of MVBs marker CD63 (green) and lysosomal marker LAMP2 (red) co-localization in cortical layers 5 ( a ) and hippocampal CA 1 - 3 ( b ) regions of WT, KIBRA −/− , 5XFAD, and KIBRA −/− 5XFAD mice. Nuclei were stained with DAPI. Scale bar in left panel = 20 µm. Scale bar in right panel = 10 µm. ( c ) Quantification analysis of overlap coefficient per view ( n = 7 in the cortex and n = 5 in the hippocampus). ( d ) WB analysis of lysosomal marker Lamp2 and Cathepsin D levels in brain tissue lysates from cortical and hippocampal regions of 3–5-month-old mice. ( e ) Quantification analysis of lysosomal membrane protein levels (Lamp2, left panel) and ratio of CatD/proCatD (right panel) in cortical and hippocampal regions from 3–5-month-old mice of the indicated genotypes. Quantification results were plotted as dot plots, showing the mean ± SE of at least three independent experiments. * P < 0.05, ⁎⁎ P < 0.01, ⁎⁎⁎⁎ P < 0.0001 as determined by a two-tailed t test. ( f ) Confocal microscopy analysis of lysosomal membrane marker Lamp2 (red) with Aβ/AβPP (anti-Aβ, clone 4G8, green) in brain slice of 5XFAD and KIBRA −/− 5XFAD mice. Scale bar = 10 µm. The overlap coefficient per cell was quantified ( n = 15). ( g ) Confocal microscopy analysis of Cathepsin D (red) with Aβ/AβPP (anti-Aβ, clone 4G8, green) in brain slice of 5XFAD and KIBRA −/− 5XFAD mice. Scale bar = 10 µm. Overlap coefficient per cell was quantified ( n = 15). f-g . Quantification result were plotted as dot plots, showing the mean ± SE. ⁎⁎⁎ P < 0.001, ⁎⁎⁎⁎ P < 0.0001 as determined by a two-tailed student's t test.

    Journal: EBioMedicine

    Article Title: KIBRA regulates amyloid β metabolism by controlling extracellular vesicles secretion

    doi: 10.1016/j.ebiom.2022.103980

    Figure Lengend Snippet: KIBRA Knockout leads to AβPP/Aβ aggregation and degradation by lysosomes. ( a and b ) Confocal microscopy analysis of MVBs marker CD63 (green) and lysosomal marker LAMP2 (red) co-localization in cortical layers 5 ( a ) and hippocampal CA 1 - 3 ( b ) regions of WT, KIBRA −/− , 5XFAD, and KIBRA −/− 5XFAD mice. Nuclei were stained with DAPI. Scale bar in left panel = 20 µm. Scale bar in right panel = 10 µm. ( c ) Quantification analysis of overlap coefficient per view ( n = 7 in the cortex and n = 5 in the hippocampus). ( d ) WB analysis of lysosomal marker Lamp2 and Cathepsin D levels in brain tissue lysates from cortical and hippocampal regions of 3–5-month-old mice. ( e ) Quantification analysis of lysosomal membrane protein levels (Lamp2, left panel) and ratio of CatD/proCatD (right panel) in cortical and hippocampal regions from 3–5-month-old mice of the indicated genotypes. Quantification results were plotted as dot plots, showing the mean ± SE of at least three independent experiments. * P < 0.05, ⁎⁎ P < 0.01, ⁎⁎⁎⁎ P < 0.0001 as determined by a two-tailed t test. ( f ) Confocal microscopy analysis of lysosomal membrane marker Lamp2 (red) with Aβ/AβPP (anti-Aβ, clone 4G8, green) in brain slice of 5XFAD and KIBRA −/− 5XFAD mice. Scale bar = 10 µm. The overlap coefficient per cell was quantified ( n = 15). ( g ) Confocal microscopy analysis of Cathepsin D (red) with Aβ/AβPP (anti-Aβ, clone 4G8, green) in brain slice of 5XFAD and KIBRA −/− 5XFAD mice. Scale bar = 10 µm. Overlap coefficient per cell was quantified ( n = 15). f-g . Quantification result were plotted as dot plots, showing the mean ± SE. ⁎⁎⁎ P < 0.001, ⁎⁎⁎⁎ P < 0.0001 as determined by a two-tailed student's t test.

    Article Snippet: The primary antibodies were obtained from the following sources: mouse monoclonal anti-KIBRA (clone 2A5, provided by Prof Jixin Dong's lab), as previously described ; mouse monoclonal anti-Alix (Cell Signaling Technology Cat# 2171; Research Resource Identifiers [RRID]: AB_2299455, Danvers, MA, USA; 1:1000 in western blot [WB]); rabbit monoclonal anti-CD63 (Abcam Cat# ab217345; RRID: AB_2754982, Cambridge, UK; 1:1000 in WB and 1:100 in immunofluorescence [IF]); rabbit polyclonal anti-CD63 (Abcam Cat# ab216130, RRID not available; 1:25 in IF); mouse monoclonal anti-APP C1/6.1 C-terminal fragment (CTFs) (Biolegend Cat# 802803, RRID: AB_2715853, San Diego, CA, USA; 1:2000 in WB); rabbit monoclonal anti-BACE1 (Abcam Cat# ab108394, RRID: AB_10861218; 1:1000 in WB); Alexa Fluor 594 anti-4G8 (Biolegend Cat# 800715, RRID: AB_2721291; 1:100 in IF); mouse monoclonal anti-4G8 (Biolegend Cat# 800712, RRID: AB_2734548; 1:400 in IF); mouse monoclonal anti-6E10 (Biolegend Cat# 803004, RRID: AB_2715854; 1:1000 in WB); rabbit polyclonal anti-calnexin (Abcam Cat# ab22595, RRID: AB_2069006; 1:1000 in WB); rabbit monoclonal anti-CD9 (Abcam Cat# ab92726, RRID: AB_10561589; 1:1000 in WB); rabbit monoclonal anti-Cathepsin D (Abcam Cat# ab75852, RRID: AB_1523267; 1:1000 in WB and 1:100 in IF); rabbit monoclonal anti-Rab7 (Abcam Cat# ab137029, RRID: AB_2629474; 1:1000 in WB); rabbit polyclonal anti-oligomer A11 (Thermo Fisher Scientific Cat# AHB0052, RRID: AB_2536236; Waltham, MA, USA; 1:1000 in Dot blots); rat monoclonal anti-LAMP2 (Abcam Cat# ab13524, RRID: AB_2134736; 1:1000 in WB and 1:100 in IF); rabbit monoclonal anti-beta III Tubulin (Abcam Cat# ab18207; RRID: AB_ 444319; 1:300 in IF); rabbit monoclonal anti-NeuN (Abcam Cat# ab177487, RRID: AB_2532109; 1:200 in IF).

    Techniques: Knock-Out, Confocal Microscopy, Marker, Staining, Two Tailed Test, Slice Preparation

    Inhibition of lysosome function in KIBRA knockout cells rescues secretion of EVs harboring APP-CTFβ/Aβ. ( a ) WB analysis of WCLs in KIBRA-KD and CTRL cells overexpressing APP swe , and treated with 20 nM bafilomycinA1 (BafA1) for 12 h, when indicated, and WCLs were blotted for Alix, CD63, and CD9. ( b ) Representative NTA traces of EVs derived from KIBRA-KD and CTRL cells overexpressing APP swe , and treated with BafA1. Right panel represents quantification of NTA of three independent experiments. ( c ) Human Aβ40 and Aβ42 levels measured by Simoa analysis from small EVs purified from sucrose step gradient centrifugation from equal number of KIBRA-KD and CTRL cells overexpressing APP swe, and treated with BafA1. ( n = 3, from three independent experiments). ( d ) WB analysis of EVs secretion in KIBRA-KD and CTRL cells overexpressing APP swe , and treated with 20 nM bafilomycinA1 (BafA1) for 12 h, when indicated. EVs were blotted for Alix, CD63, CD9, and APP-CTFβ. ( e ) Quantification of EVs protein levels in the small EVs obtained from KIBRA-KD and CTRL cells overexpressing APP swe , and treated with lysosome inhibitor BafA1 in three independent experiments. ( f ) Quantification of APP-CTFβ levels in the small EVs obtained from KIBRA-KD and CTRL cells overexpressing APP swe , and treated with lysosome inhibitor BafA1 in three independent experiments. ( g and h ) WB analysis of EVs secretion in KIBRA-KD and CTRL cells overexpressing APP swe transfected with control small interfering RNAs (siRNAs) or siRNAs targeting Rab7. WCLs ( g ) and EVs ( h ) were blotted for Alix, CD63, CD9, Rab7, and APP-CTFβ. ( i ) Quantification of EVs protein levels (Alix, CD63, and CD9) in the small EVs obtained from KIBRA-KD and CTRL cells overexpressing APP swe , and transfected with siRNAs in four independent experiments. ( j ) Quantification of APP-CTFβ levels in the small EVs obtained from KIBRA-KD and CTRL cells overexpressing APP swe , and transfected with siRNAs in three independent experiments. ( k ) Representative NTA traces of EVs derived from KIBRA-KD and CTRL cells overexpressing APP swe , and treated with siRNAs targeting Rab7. Right panel represents quantification of NTA of three independent experiments. ( l ) Human Aβ40 and Aβ42 levels measured by Simoa analysis from small EVs purified from sucrose step gradient centrifugation from equal numbers of KIBRA-KD and CTRL cells overexpressing APP swe , and treated with siRNAs in three independent experiments. The quantification results were plotted as dot plots, showing the mean ± SE. * P < 0.05, ⁎⁎ P < 0.01, ⁎⁎⁎ P < 0.001, ⁎⁎⁎⁎ P < 0.0001, n.s., not significant ( P > 0.05) as determined by one-way ANOVA followed by Tukey's post hoc comparisons tests.

    Journal: EBioMedicine

    Article Title: KIBRA regulates amyloid β metabolism by controlling extracellular vesicles secretion

    doi: 10.1016/j.ebiom.2022.103980

    Figure Lengend Snippet: Inhibition of lysosome function in KIBRA knockout cells rescues secretion of EVs harboring APP-CTFβ/Aβ. ( a ) WB analysis of WCLs in KIBRA-KD and CTRL cells overexpressing APP swe , and treated with 20 nM bafilomycinA1 (BafA1) for 12 h, when indicated, and WCLs were blotted for Alix, CD63, and CD9. ( b ) Representative NTA traces of EVs derived from KIBRA-KD and CTRL cells overexpressing APP swe , and treated with BafA1. Right panel represents quantification of NTA of three independent experiments. ( c ) Human Aβ40 and Aβ42 levels measured by Simoa analysis from small EVs purified from sucrose step gradient centrifugation from equal number of KIBRA-KD and CTRL cells overexpressing APP swe, and treated with BafA1. ( n = 3, from three independent experiments). ( d ) WB analysis of EVs secretion in KIBRA-KD and CTRL cells overexpressing APP swe , and treated with 20 nM bafilomycinA1 (BafA1) for 12 h, when indicated. EVs were blotted for Alix, CD63, CD9, and APP-CTFβ. ( e ) Quantification of EVs protein levels in the small EVs obtained from KIBRA-KD and CTRL cells overexpressing APP swe , and treated with lysosome inhibitor BafA1 in three independent experiments. ( f ) Quantification of APP-CTFβ levels in the small EVs obtained from KIBRA-KD and CTRL cells overexpressing APP swe , and treated with lysosome inhibitor BafA1 in three independent experiments. ( g and h ) WB analysis of EVs secretion in KIBRA-KD and CTRL cells overexpressing APP swe transfected with control small interfering RNAs (siRNAs) or siRNAs targeting Rab7. WCLs ( g ) and EVs ( h ) were blotted for Alix, CD63, CD9, Rab7, and APP-CTFβ. ( i ) Quantification of EVs protein levels (Alix, CD63, and CD9) in the small EVs obtained from KIBRA-KD and CTRL cells overexpressing APP swe , and transfected with siRNAs in four independent experiments. ( j ) Quantification of APP-CTFβ levels in the small EVs obtained from KIBRA-KD and CTRL cells overexpressing APP swe , and transfected with siRNAs in three independent experiments. ( k ) Representative NTA traces of EVs derived from KIBRA-KD and CTRL cells overexpressing APP swe , and treated with siRNAs targeting Rab7. Right panel represents quantification of NTA of three independent experiments. ( l ) Human Aβ40 and Aβ42 levels measured by Simoa analysis from small EVs purified from sucrose step gradient centrifugation from equal numbers of KIBRA-KD and CTRL cells overexpressing APP swe , and treated with siRNAs in three independent experiments. The quantification results were plotted as dot plots, showing the mean ± SE. * P < 0.05, ⁎⁎ P < 0.01, ⁎⁎⁎ P < 0.001, ⁎⁎⁎⁎ P < 0.0001, n.s., not significant ( P > 0.05) as determined by one-way ANOVA followed by Tukey's post hoc comparisons tests.

    Article Snippet: The primary antibodies were obtained from the following sources: mouse monoclonal anti-KIBRA (clone 2A5, provided by Prof Jixin Dong's lab), as previously described ; mouse monoclonal anti-Alix (Cell Signaling Technology Cat# 2171; Research Resource Identifiers [RRID]: AB_2299455, Danvers, MA, USA; 1:1000 in western blot [WB]); rabbit monoclonal anti-CD63 (Abcam Cat# ab217345; RRID: AB_2754982, Cambridge, UK; 1:1000 in WB and 1:100 in immunofluorescence [IF]); rabbit polyclonal anti-CD63 (Abcam Cat# ab216130, RRID not available; 1:25 in IF); mouse monoclonal anti-APP C1/6.1 C-terminal fragment (CTFs) (Biolegend Cat# 802803, RRID: AB_2715853, San Diego, CA, USA; 1:2000 in WB); rabbit monoclonal anti-BACE1 (Abcam Cat# ab108394, RRID: AB_10861218; 1:1000 in WB); Alexa Fluor 594 anti-4G8 (Biolegend Cat# 800715, RRID: AB_2721291; 1:100 in IF); mouse monoclonal anti-4G8 (Biolegend Cat# 800712, RRID: AB_2734548; 1:400 in IF); mouse monoclonal anti-6E10 (Biolegend Cat# 803004, RRID: AB_2715854; 1:1000 in WB); rabbit polyclonal anti-calnexin (Abcam Cat# ab22595, RRID: AB_2069006; 1:1000 in WB); rabbit monoclonal anti-CD9 (Abcam Cat# ab92726, RRID: AB_10561589; 1:1000 in WB); rabbit monoclonal anti-Cathepsin D (Abcam Cat# ab75852, RRID: AB_1523267; 1:1000 in WB and 1:100 in IF); rabbit monoclonal anti-Rab7 (Abcam Cat# ab137029, RRID: AB_2629474; 1:1000 in WB); rabbit polyclonal anti-oligomer A11 (Thermo Fisher Scientific Cat# AHB0052, RRID: AB_2536236; Waltham, MA, USA; 1:1000 in Dot blots); rat monoclonal anti-LAMP2 (Abcam Cat# ab13524, RRID: AB_2134736; 1:1000 in WB and 1:100 in IF); rabbit monoclonal anti-beta III Tubulin (Abcam Cat# ab18207; RRID: AB_ 444319; 1:300 in IF); rabbit monoclonal anti-NeuN (Abcam Cat# ab177487, RRID: AB_2532109; 1:200 in IF).

    Techniques: Inhibition, Knock-Out, Derivative Assay, Purification, Gradient Centrifugation, Transfection

    Primary antibodies used for Western blotting.

    Journal: Scientific Reports

    Article Title: Rejuvenation of mesenchymal stem cells by extracellular vesicles inhibits the elevation of reactive oxygen species

    doi: 10.1038/s41598-020-74444-8

    Figure Lengend Snippet: Primary antibodies used for Western blotting.

    Article Snippet: Human CD63 Antibody (CSB-PA006039) , Polyclonal Rabbit IgG , Cusabio Technology LLC, Houston, TX, USA , 1:1000.

    Techniques: Western Blot