anti-tubulin Search Results


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  • 99
    Millipore monoclonal anti beta tubulin antibody
    Monoclonal Anti Beta Tubulin Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 5041 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore α tubulin
    Acute expression of F3-T3 fusion induces peroxisome biogenesis through phosphorylation of Y122 of PIN4 a , Representative confocal microphotographs (maximum intensity) of immunofluorescence staining for total PIN4 (PIN4, red, left panel) and pY122-PIN4 (pPIN4, red, middle panel) in vector and HA expressing F3-T3. Right panels show higher magnification of dotted boxes. Nuclei were counterstained with DAPI (blue). Experiment was repeated independently two times with similar results. b , Maximum intensity of confocal images of double immunofluorescence staining for FGFR3 (green, middle panel) and pY122-PIN4 (red, right panel) in HA-F3-T3. Arrows indicate protein co-localization. Experiment was repeated independently two times with similar results. c , Co-immunoprecipitation from H1299 cells using PIN4 antibody. Endogenous PIN4 immunocomplexes and input (WCL) were analyzed by western blot using the indicated antibodies. Input is 10% for PEX1, PEX6, SUN2 and NUP214; 5% for SEC16A and DHX30; 2% for PIN4. d , Western blot analysis of co-immunoprecipitation of exogenous FLAG-PEX1 in HA-F3-T3. WCL: 1% for PIN4 and 10% for PEX1 and PEX6. Experiment was repeated independently four times with similar results. e , qRT-PCR for PEX1 in HA-F3-T3 and HA-vector. Data are Mean±s.d. (n=3 technical replicates) of one representative experiment out of three independent experiments performed in triplicate. f , Western blot analysis of PEX1 expression in HA transduced with F3-T3, F3-T3-K508M or the empty vector. β-actin is shown as loading control. Experiment was repeated independently three times with similar results. g , Time course analysis of F3-T3 expression in HA by western blot. <t>α-tubulin</t> is shown as loading control. Experiment was repeated independently two times with similar results. h , Quantification of protein biosynthesis by OPP incorporation measured by high-content fluorescent microscopy in HA reconstituted with PIN4-WT or PIN4-Y122F after silencing of the endogenous PIN4 and acutely transduced with F3-T3 or vector. Representative bar plots (n=4 technical replicates) from one out of three independent experiments. P: *
    α Tubulin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 23246 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti α tubulin
    KSHV ORF20 encodes three isoforms. (A) KSHV ORF20WT, a member of the UL24 family, can potentially express three isoforms: ORF20FL, starting at methionine 1 (M1) (aa 1–320), ORF20A, starting at leucine 24 (L24) (aa 1–297), and ORF20B, starting at M64 (aa 1–257). (B) Plasmid constructs express the three isoforms singly or in combination with each other as indicated. For analysis of ORF20A, either the genomic L24 start codon (ORF20WT and ORF20FLgA) or genomic L24 with an upstream methionine, indicated with M (ORF20A, ORF20AB) was used. * indicates genomic ORF20A leucine start codon; ORF20A starting with leucine was not detectable by immunoblotting. (C) Expression vectors encoding ORF20WT, individual ORF20 isoforms, or ORF20 isoforms in combination with each other were transfected into 293T cells. Lysates were prepared 24 h later, separated by Bis-Tris PAGE, and anti-myc and <t>anti-tubulin</t> immunoblotting was performed. Empty vector (EV) was included as a control. The immunoblot is representative of four independent experiments.
    Anti α Tubulin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 11558 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore anti tubulin
    KSHV ORF20 encodes three isoforms. (A) KSHV ORF20WT, a member of the UL24 family, can potentially express three isoforms: ORF20FL, starting at methionine 1 (M1) (aa 1–320), ORF20A, starting at leucine 24 (L24) (aa 1–297), and ORF20B, starting at M64 (aa 1–257). (B) Plasmid constructs express the three isoforms singly or in combination with each other as indicated. For analysis of ORF20A, either the genomic L24 start codon (ORF20WT and ORF20FLgA) or genomic L24 with an upstream methionine, indicated with M (ORF20A, ORF20AB) was used. * indicates genomic ORF20A leucine start codon; ORF20A starting with leucine was not detectable by immunoblotting. (C) Expression vectors encoding ORF20WT, individual ORF20 isoforms, or ORF20 isoforms in combination with each other were transfected into 293T cells. Lysates were prepared 24 h later, separated by Bis-Tris PAGE, and anti-myc and <t>anti-tubulin</t> immunoblotting was performed. Empty vector (EV) was included as a control. The immunoblot is representative of four independent experiments.
    Anti Tubulin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 6441 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology α tubulin
    Effects of CDPs on the levels of proteins involved in signaling pathways promoting mouse melanoma growth. Tumor tissue was disrupted, and cell-free protein extracts were used for Western blot assays as described in Materials and Methods. Images correspond to a representative Western blot assay ( n = 3) using protein extracts of at least three tumors homogenized from the mentioned mouse groups, using the indicated antibodies. (A) Antibodies recognized p-Akt-S473, Akt, p-mTOR-S2448, mTOR, pS6K-T389, S6K, XIAP, PDK1, NFκB p65, TNF-α, and β-actin. (B) Antibodies used were against CD44, Oct3/4, C-Myc, Ras, SNAIL, MMP-1, E-CAD, VIM, CK-1, and <t>α-tubulin.</t> On the right, graphs correspond to determination of the band intensity from the Western blot assay (left), analyzed by densitometry using the ImageJ software. Data represent the means ± SE of densitometry determinations, n = 3 for each antibody, using protein extracts obtained from tumors of each group vs. load control (β-actin or α-tubulin). One-way ANOVA with Bonferroni post-hoc test was used to compare treatments with respect to Tumor group. Significant differences ( P
    α Tubulin, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 99/100, based on 7178 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore mouse anti α tubulin
    Effects of CDPs on the levels of proteins involved in signaling pathways promoting mouse melanoma growth. Tumor tissue was disrupted, and cell-free protein extracts were used for Western blot assays as described in Materials and Methods. Images correspond to a representative Western blot assay ( n = 3) using protein extracts of at least three tumors homogenized from the mentioned mouse groups, using the indicated antibodies. (A) Antibodies recognized p-Akt-S473, Akt, p-mTOR-S2448, mTOR, pS6K-T389, S6K, XIAP, PDK1, NFκB p65, TNF-α, and β-actin. (B) Antibodies used were against CD44, Oct3/4, C-Myc, Ras, SNAIL, MMP-1, E-CAD, VIM, CK-1, and <t>α-tubulin.</t> On the right, graphs correspond to determination of the band intensity from the Western blot assay (left), analyzed by densitometry using the ImageJ software. Data represent the means ± SE of densitometry determinations, n = 3 for each antibody, using protein extracts obtained from tumors of each group vs. load control (β-actin or α-tubulin). One-way ANOVA with Bonferroni post-hoc test was used to compare treatments with respect to Tumor group. Significant differences ( P
    Mouse Anti α Tubulin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 5918 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti gamma tubulin antibody
    Effects of CDPs on the levels of proteins involved in signaling pathways promoting mouse melanoma growth. Tumor tissue was disrupted, and cell-free protein extracts were used for Western blot assays as described in Materials and Methods. Images correspond to a representative Western blot assay ( n = 3) using protein extracts of at least three tumors homogenized from the mentioned mouse groups, using the indicated antibodies. (A) Antibodies recognized p-Akt-S473, Akt, p-mTOR-S2448, mTOR, pS6K-T389, S6K, XIAP, PDK1, NFκB p65, TNF-α, and β-actin. (B) Antibodies used were against CD44, Oct3/4, C-Myc, Ras, SNAIL, MMP-1, E-CAD, VIM, CK-1, and <t>α-tubulin.</t> On the right, graphs correspond to determination of the band intensity from the Western blot assay (left), analyzed by densitometry using the ImageJ software. Data represent the means ± SE of densitometry determinations, n = 3 for each antibody, using protein extracts obtained from tumors of each group vs. load control (β-actin or α-tubulin). One-way ANOVA with Bonferroni post-hoc test was used to compare treatments with respect to Tumor group. Significant differences ( P
    Anti Gamma Tubulin Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1247 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc α tubulin
    Effects of CDPs on the levels of proteins involved in signaling pathways promoting mouse melanoma growth. Tumor tissue was disrupted, and cell-free protein extracts were used for Western blot assays as described in Materials and Methods. Images correspond to a representative Western blot assay ( n = 3) using protein extracts of at least three tumors homogenized from the mentioned mouse groups, using the indicated antibodies. (A) Antibodies recognized p-Akt-S473, Akt, p-mTOR-S2448, mTOR, pS6K-T389, S6K, XIAP, PDK1, NFκB p65, TNF-α, and β-actin. (B) Antibodies used were against CD44, Oct3/4, C-Myc, Ras, SNAIL, MMP-1, E-CAD, VIM, CK-1, and <t>α-tubulin.</t> On the right, graphs correspond to determination of the band intensity from the Western blot assay (left), analyzed by densitometry using the ImageJ software. Data represent the means ± SE of densitometry determinations, n = 3 for each antibody, using protein extracts obtained from tumors of each group vs. load control (β-actin or α-tubulin). One-way ANOVA with Bonferroni post-hoc test was used to compare treatments with respect to Tumor group. Significant differences ( P
    α Tubulin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 5143 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore monoclonal anti tubulin acetylated antibody
    Effects of CDPs on the levels of proteins involved in signaling pathways promoting mouse melanoma growth. Tumor tissue was disrupted, and cell-free protein extracts were used for Western blot assays as described in Materials and Methods. Images correspond to a representative Western blot assay ( n = 3) using protein extracts of at least three tumors homogenized from the mentioned mouse groups, using the indicated antibodies. (A) Antibodies recognized p-Akt-S473, Akt, p-mTOR-S2448, mTOR, pS6K-T389, S6K, XIAP, PDK1, NFκB p65, TNF-α, and β-actin. (B) Antibodies used were against CD44, Oct3/4, C-Myc, Ras, SNAIL, MMP-1, E-CAD, VIM, CK-1, and <t>α-tubulin.</t> On the right, graphs correspond to determination of the band intensity from the Western blot assay (left), analyzed by densitometry using the ImageJ software. Data represent the means ± SE of densitometry determinations, n = 3 for each antibody, using protein extracts obtained from tumors of each group vs. load control (β-actin or α-tubulin). One-way ANOVA with Bonferroni post-hoc test was used to compare treatments with respect to Tumor group. Significant differences ( P
    Monoclonal Anti Tubulin Acetylated Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 3808 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore monoclonal anti gamma tubulin antibody produced in mouse
    Effects of CDPs on the levels of proteins involved in signaling pathways promoting mouse melanoma growth. Tumor tissue was disrupted, and cell-free protein extracts were used for Western blot assays as described in Materials and Methods. Images correspond to a representative Western blot assay ( n = 3) using protein extracts of at least three tumors homogenized from the mentioned mouse groups, using the indicated antibodies. (A) Antibodies recognized p-Akt-S473, Akt, p-mTOR-S2448, mTOR, pS6K-T389, S6K, XIAP, PDK1, NFκB p65, TNF-α, and β-actin. (B) Antibodies used were against CD44, Oct3/4, C-Myc, Ras, SNAIL, MMP-1, E-CAD, VIM, CK-1, and <t>α-tubulin.</t> On the right, graphs correspond to determination of the band intensity from the Western blot assay (left), analyzed by densitometry using the ImageJ software. Data represent the means ± SE of densitometry determinations, n = 3 for each antibody, using protein extracts obtained from tumors of each group vs. load control (β-actin or α-tubulin). One-way ANOVA with Bonferroni post-hoc test was used to compare treatments with respect to Tumor group. Significant differences ( P
    Monoclonal Anti Gamma Tubulin Antibody Produced In Mouse, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology β tubulin
    Effects of CDPs on the levels of proteins involved in signaling pathways promoting mouse melanoma growth. Tumor tissue was disrupted, and cell-free protein extracts were used for Western blot assays as described in Materials and Methods. Images correspond to a representative Western blot assay ( n = 3) using protein extracts of at least three tumors homogenized from the mentioned mouse groups, using the indicated antibodies. (A) Antibodies recognized p-Akt-S473, Akt, p-mTOR-S2448, mTOR, pS6K-T389, S6K, XIAP, PDK1, NFκB p65, TNF-α, and β-actin. (B) Antibodies used were against CD44, Oct3/4, C-Myc, Ras, SNAIL, MMP-1, E-CAD, VIM, CK-1, and <t>α-tubulin.</t> On the right, graphs correspond to determination of the band intensity from the Western blot assay (left), analyzed by densitometry using the ImageJ software. Data represent the means ± SE of densitometry determinations, n = 3 for each antibody, using protein extracts obtained from tumors of each group vs. load control (β-actin or α-tubulin). One-way ANOVA with Bonferroni post-hoc test was used to compare treatments with respect to Tumor group. Significant differences ( P
    β Tubulin, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 97/100, based on 4138 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam alpha tubulin
    Effects of CDPs on the levels of proteins involved in signaling pathways promoting mouse melanoma growth. Tumor tissue was disrupted, and cell-free protein extracts were used for Western blot assays as described in Materials and Methods. Images correspond to a representative Western blot assay ( n = 3) using protein extracts of at least three tumors homogenized from the mentioned mouse groups, using the indicated antibodies. (A) Antibodies recognized p-Akt-S473, Akt, p-mTOR-S2448, mTOR, pS6K-T389, S6K, XIAP, PDK1, NFκB p65, TNF-α, and β-actin. (B) Antibodies used were against CD44, Oct3/4, C-Myc, Ras, SNAIL, MMP-1, E-CAD, VIM, CK-1, and <t>α-tubulin.</t> On the right, graphs correspond to determination of the band intensity from the Western blot assay (left), analyzed by densitometry using the ImageJ software. Data represent the means ± SE of densitometry determinations, n = 3 for each antibody, using protein extracts obtained from tumors of each group vs. load control (β-actin or α-tubulin). One-way ANOVA with Bonferroni post-hoc test was used to compare treatments with respect to Tumor group. Significant differences ( P
    Alpha Tubulin, supplied by Abcam, used in various techniques. Bioz Stars score: 96/100, based on 965 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam α tubulin
    Effects of CDPs on the levels of proteins involved in signaling pathways promoting mouse melanoma growth. Tumor tissue was disrupted, and cell-free protein extracts were used for Western blot assays as described in Materials and Methods. Images correspond to a representative Western blot assay ( n = 3) using protein extracts of at least three tumors homogenized from the mentioned mouse groups, using the indicated antibodies. (A) Antibodies recognized p-Akt-S473, Akt, p-mTOR-S2448, mTOR, pS6K-T389, S6K, XIAP, PDK1, NFκB p65, TNF-α, and β-actin. (B) Antibodies used were against CD44, Oct3/4, C-Myc, Ras, SNAIL, MMP-1, E-CAD, VIM, CK-1, and <t>α-tubulin.</t> On the right, graphs correspond to determination of the band intensity from the Western blot assay (left), analyzed by densitometry using the ImageJ software. Data represent the means ± SE of densitometry determinations, n = 3 for each antibody, using protein extracts obtained from tumors of each group vs. load control (β-actin or α-tubulin). One-way ANOVA with Bonferroni post-hoc test was used to compare treatments with respect to Tumor group. Significant differences ( P
    α Tubulin, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 4261 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore monoclonal anti acetylated tubulin antibody
    Reduced centrosomal <t>γ-tubulin</t> staining is specifically induced by VPS4 at the centrosome and is unaffected by ESCRT III depletion. ( a ) NIH3T3 cells were transfected with one of the indicated plasmids or siRNA construct. Fixed cells were immunostained for γ-tubulin (blue) and imaged using 3D SIM. Maximum intensity projections of representative cells are shown. GFP n = 20, scRNA n = 17, GFP- VPS4 EQ n = 21, siVPS4A/B n = 13, VPS4 EQΔMIT n = 11, siCHMP2A n = 20, siCHMP2B n = 12 and siCHMP4B n = 20. Data for each condition was obtained from at least two independent experiments. Scale, 0.2 μm. ( b ) Cells were transfected with GFP-VPS4 EQ alone (upper panel) or together with ESCRT-III components (middle and bottom panels). Fixed cells were immunostained for γ-tubulin and imaged using 3D SIM. Maximum intensity projections of reconstructed images from representative cells are shown. Left to right: an overlay image of the entire cell (scale, 10 μm), zoomed in images of the centrosome (white box): ESCRT-III (red), VPS4 EQ (green) or γ-tubulin (blue) and an overlay (scale, 0.5 μm). GFP-VPS4 EQ n = 21, co-transfection with mCherry-CHMP2A n = 12, co-transfection with mCherry-CHMP4B n = 9. ( c ) 3D volume of centrosomal γ-tubulin structure was calculated in each cell using Volocity image analysis package. Statistical analysis for average volume was calculated using a one-way ANOVA. ***p- value ≤ 0.0001. ( d ). ( e , f ) NIH3T3 cells transfected with GFP or GFP-VPS4 EQ were either harvested 24 h post transfection and subjected to western blot analysis using anti-NEDD1 antibodies ( e ), or fixed and immunostained with anti-NEDD1 antibodies ( f ). Top to bottom in ( f ): an overlay image of the entire cell (scale, 5 μm), zoomed in images of the centrosome (white box): NEDD1 (red) and an overlay (scale, 0.2 μm) GFP n = 46, GFP-VPS4 EQ n = 34. ( g ) 3D volume of centrosomal NEDD1 in each cell was calculated using Volocity image analysis package. Statistical analysis for average volume was calculated using t-test. ***p- value ≤ 0.0001.
    Monoclonal Anti Acetylated Tubulin Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2829 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc β tubulin
    Representative western blotting of intercalated disc proteins and <t>β-tubulin</t> in the right ventricular myocardium from patients with AC and healthy controls. (A) Western blotting of myocardial intercalated disc proteins in the controls, and AC patients with and without DSG2 variation, along with the quantification of the protein expression; (B) western blotting of myocardial intercalated disc proteins in the controls, and AC patients with and without PKP2 variation, along with the quantification of the protein expression. DSG2, desmoglein 2; JUP, junction plakoglobin; Cx43, connexin 43; PKP2, plakophilin 2.
    β Tubulin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 3863 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore γ tubulin
    The KIF17 CLS is necessary and sufficient for ciliary localization. ( a ) Odora, MDCK II, NIH3T3, and hTERT-RPE cells expressing full length KIF17-mCit (green) were fixed and stained for acetylated <t>tubulin</t> to mark cilia (red). Top row, images of entire cells; bottom row, higher magnification of cilia in boxed areas. White arrowheads indicate distal tips of cilia. ( b ) Left, schematic of full length human KIF17. NC, neck coil; CC, coiled-coil. Right, NIH3T3 cells expressing KIF17-mCit (green) were fixed and stained for acetylated tubulin (red) to mark cilia and <t>γ-tubulin</t> (blue) to mark the basal body. ( c-g ) Schematics of truncated and mutant KIF17 constructs (left) and their localization in Odora cells (right). Cells expressing the indicated truncated or mutant KIF17 motors (green) were fixed and stained for acetylated tubulin (red in c,f,g) or the myc tag (white in d). ( h ) Odora cells expressing full length KHC-mCit or KHC fused with the wildtype or mutant versions of the KIF17 tail were fixed and stained with antibodies to acetylated tubulin (red). Scale bars throughout figure are either 10 μm for images of entire cell or 1 μm for cilia.
    γ Tubulin, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 3234 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology tubulin
    The KIF17 CLS is necessary and sufficient for ciliary localization. ( a ) Odora, MDCK II, NIH3T3, and hTERT-RPE cells expressing full length KIF17-mCit (green) were fixed and stained for acetylated <t>tubulin</t> to mark cilia (red). Top row, images of entire cells; bottom row, higher magnification of cilia in boxed areas. White arrowheads indicate distal tips of cilia. ( b ) Left, schematic of full length human KIF17. NC, neck coil; CC, coiled-coil. Right, NIH3T3 cells expressing KIF17-mCit (green) were fixed and stained for acetylated tubulin (red) to mark cilia and <t>γ-tubulin</t> (blue) to mark the basal body. ( c-g ) Schematics of truncated and mutant KIF17 constructs (left) and their localization in Odora cells (right). Cells expressing the indicated truncated or mutant KIF17 motors (green) were fixed and stained for acetylated tubulin (red in c,f,g) or the myc tag (white in d). ( h ) Odora cells expressing full length KHC-mCit or KHC fused with the wildtype or mutant versions of the KIF17 tail were fixed and stained with antibodies to acetylated tubulin (red). Scale bars throughout figure are either 10 μm for images of entire cell or 1 μm for cilia.
    Tubulin, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 96/100, based on 3376 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc tubulin
    The KIF17 CLS is necessary and sufficient for ciliary localization. ( a ) Odora, MDCK II, NIH3T3, and hTERT-RPE cells expressing full length KIF17-mCit (green) were fixed and stained for acetylated <t>tubulin</t> to mark cilia (red). Top row, images of entire cells; bottom row, higher magnification of cilia in boxed areas. White arrowheads indicate distal tips of cilia. ( b ) Left, schematic of full length human KIF17. NC, neck coil; CC, coiled-coil. Right, NIH3T3 cells expressing KIF17-mCit (green) were fixed and stained for acetylated tubulin (red) to mark cilia and <t>γ-tubulin</t> (blue) to mark the basal body. ( c-g ) Schematics of truncated and mutant KIF17 constructs (left) and their localization in Odora cells (right). Cells expressing the indicated truncated or mutant KIF17 motors (green) were fixed and stained for acetylated tubulin (red in c,f,g) or the myc tag (white in d). ( h ) Odora cells expressing full length KHC-mCit or KHC fused with the wildtype or mutant versions of the KIF17 tail were fixed and stained with antibodies to acetylated tubulin (red). Scale bars throughout figure are either 10 μm for images of entire cell or 1 μm for cilia.
    Tubulin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 2637 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    N/A
    TUBG2 Tubulin Gamma 2 is a Protein Coding gene Among its related pathways are PAK Pathway and PI3K Akt signaling pathway Gene Ontology GO annotations related to this gene include
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    N/A
    This gene encodes a beta tubulin protein This protein forms a dimer with alpha tubulin and acts as a structural component of microtubules Mutations in this gene cause cortical dysplasia
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    N/A
    There are five tubulins in human cells alpha beta gamma delta and epsilon Tubulins are conserved across species They form heterodimers which multimerize to form a microtubule filament An alpha
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    Acute expression of F3-T3 fusion induces peroxisome biogenesis through phosphorylation of Y122 of PIN4 a , Representative confocal microphotographs (maximum intensity) of immunofluorescence staining for total PIN4 (PIN4, red, left panel) and pY122-PIN4 (pPIN4, red, middle panel) in vector and HA expressing F3-T3. Right panels show higher magnification of dotted boxes. Nuclei were counterstained with DAPI (blue). Experiment was repeated independently two times with similar results. b , Maximum intensity of confocal images of double immunofluorescence staining for FGFR3 (green, middle panel) and pY122-PIN4 (red, right panel) in HA-F3-T3. Arrows indicate protein co-localization. Experiment was repeated independently two times with similar results. c , Co-immunoprecipitation from H1299 cells using PIN4 antibody. Endogenous PIN4 immunocomplexes and input (WCL) were analyzed by western blot using the indicated antibodies. Input is 10% for PEX1, PEX6, SUN2 and NUP214; 5% for SEC16A and DHX30; 2% for PIN4. d , Western blot analysis of co-immunoprecipitation of exogenous FLAG-PEX1 in HA-F3-T3. WCL: 1% for PIN4 and 10% for PEX1 and PEX6. Experiment was repeated independently four times with similar results. e , qRT-PCR for PEX1 in HA-F3-T3 and HA-vector. Data are Mean±s.d. (n=3 technical replicates) of one representative experiment out of three independent experiments performed in triplicate. f , Western blot analysis of PEX1 expression in HA transduced with F3-T3, F3-T3-K508M or the empty vector. β-actin is shown as loading control. Experiment was repeated independently three times with similar results. g , Time course analysis of F3-T3 expression in HA by western blot. α-tubulin is shown as loading control. Experiment was repeated independently two times with similar results. h , Quantification of protein biosynthesis by OPP incorporation measured by high-content fluorescent microscopy in HA reconstituted with PIN4-WT or PIN4-Y122F after silencing of the endogenous PIN4 and acutely transduced with F3-T3 or vector. Representative bar plots (n=4 technical replicates) from one out of three independent experiments. P: *

    Journal: Nature

    Article Title: A metabolic function of FGFR3-TACC3 gene fusions in cancer

    doi: 10.1038/nature25171

    Figure Lengend Snippet: Acute expression of F3-T3 fusion induces peroxisome biogenesis through phosphorylation of Y122 of PIN4 a , Representative confocal microphotographs (maximum intensity) of immunofluorescence staining for total PIN4 (PIN4, red, left panel) and pY122-PIN4 (pPIN4, red, middle panel) in vector and HA expressing F3-T3. Right panels show higher magnification of dotted boxes. Nuclei were counterstained with DAPI (blue). Experiment was repeated independently two times with similar results. b , Maximum intensity of confocal images of double immunofluorescence staining for FGFR3 (green, middle panel) and pY122-PIN4 (red, right panel) in HA-F3-T3. Arrows indicate protein co-localization. Experiment was repeated independently two times with similar results. c , Co-immunoprecipitation from H1299 cells using PIN4 antibody. Endogenous PIN4 immunocomplexes and input (WCL) were analyzed by western blot using the indicated antibodies. Input is 10% for PEX1, PEX6, SUN2 and NUP214; 5% for SEC16A and DHX30; 2% for PIN4. d , Western blot analysis of co-immunoprecipitation of exogenous FLAG-PEX1 in HA-F3-T3. WCL: 1% for PIN4 and 10% for PEX1 and PEX6. Experiment was repeated independently four times with similar results. e , qRT-PCR for PEX1 in HA-F3-T3 and HA-vector. Data are Mean±s.d. (n=3 technical replicates) of one representative experiment out of three independent experiments performed in triplicate. f , Western blot analysis of PEX1 expression in HA transduced with F3-T3, F3-T3-K508M or the empty vector. β-actin is shown as loading control. Experiment was repeated independently three times with similar results. g , Time course analysis of F3-T3 expression in HA by western blot. α-tubulin is shown as loading control. Experiment was repeated independently two times with similar results. h , Quantification of protein biosynthesis by OPP incorporation measured by high-content fluorescent microscopy in HA reconstituted with PIN4-WT or PIN4-Y122F after silencing of the endogenous PIN4 and acutely transduced with F3-T3 or vector. Representative bar plots (n=4 technical replicates) from one out of three independent experiments. P: *

    Article Snippet: Antibodies and concentrations are: FGFR3 1:1000 (Santa Cruz, B-9, sc-13121), PIN4 1:1000 (Abcam, ab155283), PKM2 1:1000 (Cell Signaling, #3198), DLG3/SAP102 1:1000 (Cell Signaling, #3733), GOLGIN84 1:2000 (Santa Cruz, H-283, sc-134704); C1ORF50 1:1000 (Novus Biologicals, NBP1-81053), HGS 1:1000 (Abcam, ab72053), FAK 1:1000 (Cell Signaling, 3285), Paxillin 1:1000 (BD Transduction, 610051), PGC1α 1:500 (Santa Cruz, H300, sc-13067), PGC1α 1:1000 (Novus Biological, NBP104676), ESRRG 1:500 (Abcam, ab128930), ESRRG 1:500 (R7D, PP-H6812000) p-FRS2 1:1000 (Cell Signaling, 3861), FRS2 1:1000 (Santa Cruz, sc-8318), p-STAT3 1:1000 (Cell Signaling, #9131), STAT3 1:1000 (Santa Cruz, C-20 sc-482,), p-AKT 1:1000 (Cell Signaling, #4060), AKT 1:1000 (Cell Signaling, #9272), p-ERK1/2 1:1000 (Cell Signaling, #4370), ERK1/2 1:1000 (Cell Signaling, #9102), β-actin 1:2000 (Sigma, A5441), PEX1 1:500 (BD Biosciences #611719), PEX6 1:500 (Stress Marq, #SMC-470), NUP214 1:500 (Abcam #ab70497), SEC16A 1:500 (Abcam #ab70722), DHX30 1:500 (Novus Biologicals, NBP1-26203), SUN-2 1:500 (Abcam #ab124916), FLAG 1:1000 (Abcam ab1162), Retinoblastoma 1:1000 (BD Pharmingen 554136), α-tubulin 1:2000 (Sigma, T5168), total OXPHOS 1:1000 (Abcam, #ab110411), MTCO1 1:1000 (Abcam, #ab14705).

    Techniques: Expressing, Immunofluorescence, Staining, Plasmid Preparation, Double Immunofluorescence Staining, Immunoprecipitation, Western Blot, Quantitative RT-PCR, Transduction, Microscopy

    Functional analysis of tyrosine phosphorylation of F3-T3 kinase substrates a , Western blot analysis of phosphotyrosine immunoprecipitation of mGSC-F3-T3-sh TP53 and mGSC-HRAS-12V-sh TP53 using PIN4 antibody. F3-T3 and RAS-12V expression are shown. α-tubulin is shown as loading control b , Microphotographs of immunofluorescence using the pY122-PIN4 specific antibody (red, upper panels) in tumors from mGSC-F3-T3-sh TP53 and mGSC-HRAS-12V-sh TP53. Nuclei were counterstained with DAPI (blue, lower panels). Experiment was repeated independently two times with similar results. c , Representative microphotographs of pY122-PIN4 immunofluorescence in F3-T3-positive (upper panels) and F3-T3-negative (lower panels) GBM (green). Right panels show higher magnification of pY122-PIN4-DAPI co-staining depicting cytoplasmic localization of pY122-PIN4. DAPI staining of nuclei is shown as an indication of cellular density (middle panels). d , Analysis of OCR of HA-F3-T3 transduced with WT, or the unphosphorylable mutant (Y/A) of GOLGIN84, C1ORF50 and DLG3. HA-vector are included as control. Data are Mean±s.d. (n=5 technical replicates) of one representative experiment out of two independent experiments performed in triplicate with similar results. P

    Journal: Nature

    Article Title: A metabolic function of FGFR3-TACC3 gene fusions in cancer

    doi: 10.1038/nature25171

    Figure Lengend Snippet: Functional analysis of tyrosine phosphorylation of F3-T3 kinase substrates a , Western blot analysis of phosphotyrosine immunoprecipitation of mGSC-F3-T3-sh TP53 and mGSC-HRAS-12V-sh TP53 using PIN4 antibody. F3-T3 and RAS-12V expression are shown. α-tubulin is shown as loading control b , Microphotographs of immunofluorescence using the pY122-PIN4 specific antibody (red, upper panels) in tumors from mGSC-F3-T3-sh TP53 and mGSC-HRAS-12V-sh TP53. Nuclei were counterstained with DAPI (blue, lower panels). Experiment was repeated independently two times with similar results. c , Representative microphotographs of pY122-PIN4 immunofluorescence in F3-T3-positive (upper panels) and F3-T3-negative (lower panels) GBM (green). Right panels show higher magnification of pY122-PIN4-DAPI co-staining depicting cytoplasmic localization of pY122-PIN4. DAPI staining of nuclei is shown as an indication of cellular density (middle panels). d , Analysis of OCR of HA-F3-T3 transduced with WT, or the unphosphorylable mutant (Y/A) of GOLGIN84, C1ORF50 and DLG3. HA-vector are included as control. Data are Mean±s.d. (n=5 technical replicates) of one representative experiment out of two independent experiments performed in triplicate with similar results. P

    Article Snippet: Antibodies and concentrations are: FGFR3 1:1000 (Santa Cruz, B-9, sc-13121), PIN4 1:1000 (Abcam, ab155283), PKM2 1:1000 (Cell Signaling, #3198), DLG3/SAP102 1:1000 (Cell Signaling, #3733), GOLGIN84 1:2000 (Santa Cruz, H-283, sc-134704); C1ORF50 1:1000 (Novus Biologicals, NBP1-81053), HGS 1:1000 (Abcam, ab72053), FAK 1:1000 (Cell Signaling, 3285), Paxillin 1:1000 (BD Transduction, 610051), PGC1α 1:500 (Santa Cruz, H300, sc-13067), PGC1α 1:1000 (Novus Biological, NBP104676), ESRRG 1:500 (Abcam, ab128930), ESRRG 1:500 (R7D, PP-H6812000) p-FRS2 1:1000 (Cell Signaling, 3861), FRS2 1:1000 (Santa Cruz, sc-8318), p-STAT3 1:1000 (Cell Signaling, #9131), STAT3 1:1000 (Santa Cruz, C-20 sc-482,), p-AKT 1:1000 (Cell Signaling, #4060), AKT 1:1000 (Cell Signaling, #9272), p-ERK1/2 1:1000 (Cell Signaling, #4370), ERK1/2 1:1000 (Cell Signaling, #9102), β-actin 1:2000 (Sigma, A5441), PEX1 1:500 (BD Biosciences #611719), PEX6 1:500 (Stress Marq, #SMC-470), NUP214 1:500 (Abcam #ab70497), SEC16A 1:500 (Abcam #ab70722), DHX30 1:500 (Novus Biologicals, NBP1-26203), SUN-2 1:500 (Abcam #ab124916), FLAG 1:1000 (Abcam ab1162), Retinoblastoma 1:1000 (BD Pharmingen 554136), α-tubulin 1:2000 (Sigma, T5168), total OXPHOS 1:1000 (Abcam, #ab110411), MTCO1 1:1000 (Abcam, #ab14705).

    Techniques: Functional Assay, Western Blot, Immunoprecipitation, Expressing, Immunofluorescence, Staining, Transduction, Mutagenesis, Plasmid Preparation

    F3-T3 induces sensitivity to inhibitors of mitochondrial metabolism a , Immunoblot analysis using the FGFR3 antibody in HA-vector, HA-F3-T3 or HA-F3-T3-K508M. α-tubulin is shown as loading control. Experiment was repeated five times with similar results. b , OCR of GSC1123 harboring F3-T3 in the presence or absence of AZD4547. Data are Mean±s.d. (n=6 technical replicates) of one representative experiment out of two independent experiments. P:

    Journal: Nature

    Article Title: A metabolic function of FGFR3-TACC3 gene fusions in cancer

    doi: 10.1038/nature25171

    Figure Lengend Snippet: F3-T3 induces sensitivity to inhibitors of mitochondrial metabolism a , Immunoblot analysis using the FGFR3 antibody in HA-vector, HA-F3-T3 or HA-F3-T3-K508M. α-tubulin is shown as loading control. Experiment was repeated five times with similar results. b , OCR of GSC1123 harboring F3-T3 in the presence or absence of AZD4547. Data are Mean±s.d. (n=6 technical replicates) of one representative experiment out of two independent experiments. P:

    Article Snippet: Antibodies and concentrations are: FGFR3 1:1000 (Santa Cruz, B-9, sc-13121), PIN4 1:1000 (Abcam, ab155283), PKM2 1:1000 (Cell Signaling, #3198), DLG3/SAP102 1:1000 (Cell Signaling, #3733), GOLGIN84 1:2000 (Santa Cruz, H-283, sc-134704); C1ORF50 1:1000 (Novus Biologicals, NBP1-81053), HGS 1:1000 (Abcam, ab72053), FAK 1:1000 (Cell Signaling, 3285), Paxillin 1:1000 (BD Transduction, 610051), PGC1α 1:500 (Santa Cruz, H300, sc-13067), PGC1α 1:1000 (Novus Biological, NBP104676), ESRRG 1:500 (Abcam, ab128930), ESRRG 1:500 (R7D, PP-H6812000) p-FRS2 1:1000 (Cell Signaling, 3861), FRS2 1:1000 (Santa Cruz, sc-8318), p-STAT3 1:1000 (Cell Signaling, #9131), STAT3 1:1000 (Santa Cruz, C-20 sc-482,), p-AKT 1:1000 (Cell Signaling, #4060), AKT 1:1000 (Cell Signaling, #9272), p-ERK1/2 1:1000 (Cell Signaling, #4370), ERK1/2 1:1000 (Cell Signaling, #9102), β-actin 1:2000 (Sigma, A5441), PEX1 1:500 (BD Biosciences #611719), PEX6 1:500 (Stress Marq, #SMC-470), NUP214 1:500 (Abcam #ab70497), SEC16A 1:500 (Abcam #ab70722), DHX30 1:500 (Novus Biologicals, NBP1-26203), SUN-2 1:500 (Abcam #ab124916), FLAG 1:1000 (Abcam ab1162), Retinoblastoma 1:1000 (BD Pharmingen 554136), α-tubulin 1:2000 (Sigma, T5168), total OXPHOS 1:1000 (Abcam, #ab110411), MTCO1 1:1000 (Abcam, #ab14705).

    Techniques: Plasmid Preparation

    KSHV ORF20 encodes three isoforms. (A) KSHV ORF20WT, a member of the UL24 family, can potentially express three isoforms: ORF20FL, starting at methionine 1 (M1) (aa 1–320), ORF20A, starting at leucine 24 (L24) (aa 1–297), and ORF20B, starting at M64 (aa 1–257). (B) Plasmid constructs express the three isoforms singly or in combination with each other as indicated. For analysis of ORF20A, either the genomic L24 start codon (ORF20WT and ORF20FLgA) or genomic L24 with an upstream methionine, indicated with M (ORF20A, ORF20AB) was used. * indicates genomic ORF20A leucine start codon; ORF20A starting with leucine was not detectable by immunoblotting. (C) Expression vectors encoding ORF20WT, individual ORF20 isoforms, or ORF20 isoforms in combination with each other were transfected into 293T cells. Lysates were prepared 24 h later, separated by Bis-Tris PAGE, and anti-myc and anti-tubulin immunoblotting was performed. Empty vector (EV) was included as a control. The immunoblot is representative of four independent experiments.

    Journal: PLoS Pathogens

    Article Title: The interferon-stimulated gene product oligoadenylate synthetase-like protein enhances replication of Kaposi’s sarcoma-associated herpesvirus (KSHV) and interacts with the KSHV ORF20 protein

    doi: 10.1371/journal.ppat.1006937

    Figure Lengend Snippet: KSHV ORF20 encodes three isoforms. (A) KSHV ORF20WT, a member of the UL24 family, can potentially express three isoforms: ORF20FL, starting at methionine 1 (M1) (aa 1–320), ORF20A, starting at leucine 24 (L24) (aa 1–297), and ORF20B, starting at M64 (aa 1–257). (B) Plasmid constructs express the three isoforms singly or in combination with each other as indicated. For analysis of ORF20A, either the genomic L24 start codon (ORF20WT and ORF20FLgA) or genomic L24 with an upstream methionine, indicated with M (ORF20A, ORF20AB) was used. * indicates genomic ORF20A leucine start codon; ORF20A starting with leucine was not detectable by immunoblotting. (C) Expression vectors encoding ORF20WT, individual ORF20 isoforms, or ORF20 isoforms in combination with each other were transfected into 293T cells. Lysates were prepared 24 h later, separated by Bis-Tris PAGE, and anti-myc and anti-tubulin immunoblotting was performed. Empty vector (EV) was included as a control. The immunoblot is representative of four independent experiments.

    Article Snippet: Rabbit anti-c-myc (# C3956), mouse anti-tubulin (# T6199-200UL), and mouse anti-beta-actin (#A5441) antibodies were purchased from Sigma-Aldrich.

    Techniques: Plasmid Preparation, Construct, Expressing, Transfection, Polyacrylamide Gel Electrophoresis

    The interaction of ORF20 with OASL is conserved among the members of the UL24 family. (A) HeLa cells were transfected with the indicated plasmid and seeded onto coverslips. 48 h post transfection, coverslips were fixed in PFA and processed for anti-myc (green) and anti-fibrillarin (red) immunofluorescence. Nuclei were counterstained with Hoechst (blue). Images are representative of three independent experiments. Scale bar = 10 μm (B) 293T cells were transfected with myc-tagged UL24, UL76, M76, or ORF20WT. Lysates were prepared 24 h later, separated by SDS-PAGE, and anti-myc and anti-tubulin immunoblotting was performed. Data are representative of four independent experiments. (C) 293T cells were transfected with the indicated myc-tagged KSHV ORF20 form or MHV68 ORF20. An anti-myc immunoprecipitation of RIPA lysates was performed and input lysates and immunoprecipitates were immunoblotted with an anti-myc antibody. Data are representative of two independent experiments. (D) 293T cells were transfected with myc-tagged UL24 homologs MCMV M76, HCMV UL76, or HSV-1 UL24, LacZ-myc, OASL-V5, and/or EV as indicated. An anti-myc immunoprecipitation of RIPA lysates was performed and input lysates and immunoprecipitates were immunoblotted with anti-V5 and anti-myc antibodies. Data are representative of two independent experiments.

    Journal: PLoS Pathogens

    Article Title: The interferon-stimulated gene product oligoadenylate synthetase-like protein enhances replication of Kaposi’s sarcoma-associated herpesvirus (KSHV) and interacts with the KSHV ORF20 protein

    doi: 10.1371/journal.ppat.1006937

    Figure Lengend Snippet: The interaction of ORF20 with OASL is conserved among the members of the UL24 family. (A) HeLa cells were transfected with the indicated plasmid and seeded onto coverslips. 48 h post transfection, coverslips were fixed in PFA and processed for anti-myc (green) and anti-fibrillarin (red) immunofluorescence. Nuclei were counterstained with Hoechst (blue). Images are representative of three independent experiments. Scale bar = 10 μm (B) 293T cells were transfected with myc-tagged UL24, UL76, M76, or ORF20WT. Lysates were prepared 24 h later, separated by SDS-PAGE, and anti-myc and anti-tubulin immunoblotting was performed. Data are representative of four independent experiments. (C) 293T cells were transfected with the indicated myc-tagged KSHV ORF20 form or MHV68 ORF20. An anti-myc immunoprecipitation of RIPA lysates was performed and input lysates and immunoprecipitates were immunoblotted with an anti-myc antibody. Data are representative of two independent experiments. (D) 293T cells were transfected with myc-tagged UL24 homologs MCMV M76, HCMV UL76, or HSV-1 UL24, LacZ-myc, OASL-V5, and/or EV as indicated. An anti-myc immunoprecipitation of RIPA lysates was performed and input lysates and immunoprecipitates were immunoblotted with anti-V5 and anti-myc antibodies. Data are representative of two independent experiments.

    Article Snippet: Rabbit anti-c-myc (# C3956), mouse anti-tubulin (# T6199-200UL), and mouse anti-beta-actin (#A5441) antibodies were purchased from Sigma-Aldrich.

    Techniques: Transfection, Plasmid Preparation, Immunofluorescence, SDS Page, Immunoprecipitation

    Effects of CDPs on the levels of proteins involved in signaling pathways promoting mouse melanoma growth. Tumor tissue was disrupted, and cell-free protein extracts were used for Western blot assays as described in Materials and Methods. Images correspond to a representative Western blot assay ( n = 3) using protein extracts of at least three tumors homogenized from the mentioned mouse groups, using the indicated antibodies. (A) Antibodies recognized p-Akt-S473, Akt, p-mTOR-S2448, mTOR, pS6K-T389, S6K, XIAP, PDK1, NFκB p65, TNF-α, and β-actin. (B) Antibodies used were against CD44, Oct3/4, C-Myc, Ras, SNAIL, MMP-1, E-CAD, VIM, CK-1, and α-tubulin. On the right, graphs correspond to determination of the band intensity from the Western blot assay (left), analyzed by densitometry using the ImageJ software. Data represent the means ± SE of densitometry determinations, n = 3 for each antibody, using protein extracts obtained from tumors of each group vs. load control (β-actin or α-tubulin). One-way ANOVA with Bonferroni post-hoc test was used to compare treatments with respect to Tumor group. Significant differences ( P

    Journal: Frontiers in Oncology

    Article Title: Bacterial Cyclodipeptides Target Signal Pathways Involved in Malignant Melanoma

    doi: 10.3389/fonc.2020.01111

    Figure Lengend Snippet: Effects of CDPs on the levels of proteins involved in signaling pathways promoting mouse melanoma growth. Tumor tissue was disrupted, and cell-free protein extracts were used for Western blot assays as described in Materials and Methods. Images correspond to a representative Western blot assay ( n = 3) using protein extracts of at least three tumors homogenized from the mentioned mouse groups, using the indicated antibodies. (A) Antibodies recognized p-Akt-S473, Akt, p-mTOR-S2448, mTOR, pS6K-T389, S6K, XIAP, PDK1, NFκB p65, TNF-α, and β-actin. (B) Antibodies used were against CD44, Oct3/4, C-Myc, Ras, SNAIL, MMP-1, E-CAD, VIM, CK-1, and α-tubulin. On the right, graphs correspond to determination of the band intensity from the Western blot assay (left), analyzed by densitometry using the ImageJ software. Data represent the means ± SE of densitometry determinations, n = 3 for each antibody, using protein extracts obtained from tumors of each group vs. load control (β-actin or α-tubulin). One-way ANOVA with Bonferroni post-hoc test was used to compare treatments with respect to Tumor group. Significant differences ( P

    Article Snippet: Polyvinylidene difluoride membranes were cut according to range of molecular weight markers and incubated with the indicated antibodies at the concentration suggested by the manufacturer: anti-CD44, anti-Oct3/4, anti-C-Myc, anti-Ras, anti-SNAIL, anti-MMP-1, anti-E-Cad, anti-vimentin, anti-cytokeratin 1 (CK-1), anti-α-tubulin, anti-Akt (C-20), anti-Akt-phosphorylated 1/2/3 (S-473), anti-mTOR, anti-phosphorylated-mTOR-(S2448), anti-β-actin, and anti-α-tubulin (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibodies.

    Techniques: Western Blot, Software

    Reduced centrosomal γ-tubulin staining is specifically induced by VPS4 at the centrosome and is unaffected by ESCRT III depletion. ( a ) NIH3T3 cells were transfected with one of the indicated plasmids or siRNA construct. Fixed cells were immunostained for γ-tubulin (blue) and imaged using 3D SIM. Maximum intensity projections of representative cells are shown. GFP n = 20, scRNA n = 17, GFP- VPS4 EQ n = 21, siVPS4A/B n = 13, VPS4 EQΔMIT n = 11, siCHMP2A n = 20, siCHMP2B n = 12 and siCHMP4B n = 20. Data for each condition was obtained from at least two independent experiments. Scale, 0.2 μm. ( b ) Cells were transfected with GFP-VPS4 EQ alone (upper panel) or together with ESCRT-III components (middle and bottom panels). Fixed cells were immunostained for γ-tubulin and imaged using 3D SIM. Maximum intensity projections of reconstructed images from representative cells are shown. Left to right: an overlay image of the entire cell (scale, 10 μm), zoomed in images of the centrosome (white box): ESCRT-III (red), VPS4 EQ (green) or γ-tubulin (blue) and an overlay (scale, 0.5 μm). GFP-VPS4 EQ n = 21, co-transfection with mCherry-CHMP2A n = 12, co-transfection with mCherry-CHMP4B n = 9. ( c ) 3D volume of centrosomal γ-tubulin structure was calculated in each cell using Volocity image analysis package. Statistical analysis for average volume was calculated using a one-way ANOVA. ***p- value ≤ 0.0001. ( d ). ( e , f ) NIH3T3 cells transfected with GFP or GFP-VPS4 EQ were either harvested 24 h post transfection and subjected to western blot analysis using anti-NEDD1 antibodies ( e ), or fixed and immunostained with anti-NEDD1 antibodies ( f ). Top to bottom in ( f ): an overlay image of the entire cell (scale, 5 μm), zoomed in images of the centrosome (white box): NEDD1 (red) and an overlay (scale, 0.2 μm) GFP n = 46, GFP-VPS4 EQ n = 34. ( g ) 3D volume of centrosomal NEDD1 in each cell was calculated using Volocity image analysis package. Statistical analysis for average volume was calculated using t-test. ***p- value ≤ 0.0001.

    Journal: Scientific Reports

    Article Title: VPS4 is a dynamic component of the centrosome that regulates centrosome localization of γ-tubulin, centriolar satellite stability and ciliogenesis

    doi: 10.1038/s41598-018-21491-x

    Figure Lengend Snippet: Reduced centrosomal γ-tubulin staining is specifically induced by VPS4 at the centrosome and is unaffected by ESCRT III depletion. ( a ) NIH3T3 cells were transfected with one of the indicated plasmids or siRNA construct. Fixed cells were immunostained for γ-tubulin (blue) and imaged using 3D SIM. Maximum intensity projections of representative cells are shown. GFP n = 20, scRNA n = 17, GFP- VPS4 EQ n = 21, siVPS4A/B n = 13, VPS4 EQΔMIT n = 11, siCHMP2A n = 20, siCHMP2B n = 12 and siCHMP4B n = 20. Data for each condition was obtained from at least two independent experiments. Scale, 0.2 μm. ( b ) Cells were transfected with GFP-VPS4 EQ alone (upper panel) or together with ESCRT-III components (middle and bottom panels). Fixed cells were immunostained for γ-tubulin and imaged using 3D SIM. Maximum intensity projections of reconstructed images from representative cells are shown. Left to right: an overlay image of the entire cell (scale, 10 μm), zoomed in images of the centrosome (white box): ESCRT-III (red), VPS4 EQ (green) or γ-tubulin (blue) and an overlay (scale, 0.5 μm). GFP-VPS4 EQ n = 21, co-transfection with mCherry-CHMP2A n = 12, co-transfection with mCherry-CHMP4B n = 9. ( c ) 3D volume of centrosomal γ-tubulin structure was calculated in each cell using Volocity image analysis package. Statistical analysis for average volume was calculated using a one-way ANOVA. ***p- value ≤ 0.0001. ( d ). ( e , f ) NIH3T3 cells transfected with GFP or GFP-VPS4 EQ were either harvested 24 h post transfection and subjected to western blot analysis using anti-NEDD1 antibodies ( e ), or fixed and immunostained with anti-NEDD1 antibodies ( f ). Top to bottom in ( f ): an overlay image of the entire cell (scale, 5 μm), zoomed in images of the centrosome (white box): NEDD1 (red) and an overlay (scale, 0.2 μm) GFP n = 46, GFP-VPS4 EQ n = 34. ( g ) 3D volume of centrosomal NEDD1 in each cell was calculated using Volocity image analysis package. Statistical analysis for average volume was calculated using t-test. ***p- value ≤ 0.0001.

    Article Snippet: Cells were then permeabilized, blocked and immunostained with the following primary antibodies as specified in text: mouse monoclonal anti α-tubulin antibodies (1:1000, DM1A clone Sigma-Aldrich Cat # T9026), mouse monoclonal anti acetylated tubulin antibodies (1:500 or 1:1000, Thermo Scientific Cat # 32–2700 or 1:1000, Sigma-Aldrich Cat # T7451), mouse monoclonal anti γ-tubulin (1:1000, Sigma-Aldrich Cat #T6557), mouse monoclonal anti NEDD1 (1:100, Abcam Cat # ab57336), rabbit polyclonal anti γ-tubulin antibodies (1:200, Abcam Cat # ab 84355), rabbit polyclonal anti CHMP2A (1:50, Proteintech Group Cat # 10477-1-AP) rabbit polyclonal anti CHMP2B (1:50, Proteintech Group Cat # 12527-1-AP), rabbit polyclonal anti IST1 (1:50, Proteintech Group Cat # 51002-1-AP), rabbit polyclonal anti CP110 (1:50, Proteintech Group Cat # 12780-1-AP) antibodies, rabbit polyclonal anti CHMP4A (1:50, Santa Cruz Biotechnology Cat # SC-67229), rabbit polyclonal anti PCM1 antibodies (1:100, Santa Cruz Biotechnology, Cat # SC-67204), rabbit polyclonal anti Cep164 (1: 200, Abcam Cat # ab106372 or 1:200 Proteintech Group Cat # 22227-1-AP), rabbit polyclonal anti Cep290 (1:100, Abcam Cat # ab84870), mouse anti Centrin (1:500, Millipore clone 20H5, Cat # 04-1624) and rabbit polyclonal anti VTA1 antibodies (1:50, Pierce Biotechnology Cat # PA521831).

    Techniques: Staining, Transfection, Construct, Cotransfection, Western Blot

    ESCRT-III components do not recruit VPS4 to centrosomes. ( a ) Maximum projection images of representative NIH3T3 cells, transfected with Pericentrin–RFP, and the indicated plasmids or immunostained for the endogenous ESCRT-III proteins CHMP2A or CHMP2B are shown. Top to bottom: entire cell (scale 10 μm), zoomed-in images of the centrosome (white box): ESCRT-III component (green), Pericentrin (red), an overlay (scale, 1 μm) and a line intensity profile of both channels along the centrosome. ( b ). n > 100, from at least two independent experiments. ( c ) NIH3T3 cells expressing GFP-VPS4 EQ were fixed, immunostained with the indicated ESCRT-III antibodies and imaged using a confocal spinning disk microscope. Shown are representative images (data obtained from at least two independent experiments for each protein tested). Top to bottom: entire cell (scale, 10 μm), zoomed-in images (white box) of: ESCRT-III (red), VPS4 EQ (green) and an overlay (scale, 1 μm). ( d ) NIH3T3 cells, transfected with GFP-VPS4 EQ and the indicated ESCRT III components, were immunostained with anti γ-tubulin and imaged. Shown are images of representative cells. Upper panel: entire cells (scale, 10 μm). Zoomed-in images (marked by squares in upper panel) of MVBs (left; yellow) and the centrosome (right; white) are shown below: VPS4 EQ (green), ESCRT III (red), γ-tubulin (blue) (scale 1 μm). Co-transfection with mCherry-CHMP2A n = 12, co- transfection with mCherry-CHMP4B n = 9. ( e ) VPS4 is recruited to the centrosome independent of ESCRT III. NIH3T3 cells were co-transfected with GFP-VPS4 EQ and mCherry-CHMP6-N peptide (composed of the first 52 amino acids of CHMP6) (upper panel) or with siCHMP4B and GFP-VPS4 EQ (bottom panel). Cells were then fixed, immunostained with γ-tubulin antibodies and imaged. Left to right: entire cell (scale, 10 μm), zoomed-in images (white box) of: γ-tubulin (blue), VPS4 EQ (green) and an overlay (scale, 1 μm). White arrows mark the centrosome. CHMP6-N n = 21, siCHMP4B n = 13.

    Journal: Scientific Reports

    Article Title: VPS4 is a dynamic component of the centrosome that regulates centrosome localization of γ-tubulin, centriolar satellite stability and ciliogenesis

    doi: 10.1038/s41598-018-21491-x

    Figure Lengend Snippet: ESCRT-III components do not recruit VPS4 to centrosomes. ( a ) Maximum projection images of representative NIH3T3 cells, transfected with Pericentrin–RFP, and the indicated plasmids or immunostained for the endogenous ESCRT-III proteins CHMP2A or CHMP2B are shown. Top to bottom: entire cell (scale 10 μm), zoomed-in images of the centrosome (white box): ESCRT-III component (green), Pericentrin (red), an overlay (scale, 1 μm) and a line intensity profile of both channels along the centrosome. ( b ). n > 100, from at least two independent experiments. ( c ) NIH3T3 cells expressing GFP-VPS4 EQ were fixed, immunostained with the indicated ESCRT-III antibodies and imaged using a confocal spinning disk microscope. Shown are representative images (data obtained from at least two independent experiments for each protein tested). Top to bottom: entire cell (scale, 10 μm), zoomed-in images (white box) of: ESCRT-III (red), VPS4 EQ (green) and an overlay (scale, 1 μm). ( d ) NIH3T3 cells, transfected with GFP-VPS4 EQ and the indicated ESCRT III components, were immunostained with anti γ-tubulin and imaged. Shown are images of representative cells. Upper panel: entire cells (scale, 10 μm). Zoomed-in images (marked by squares in upper panel) of MVBs (left; yellow) and the centrosome (right; white) are shown below: VPS4 EQ (green), ESCRT III (red), γ-tubulin (blue) (scale 1 μm). Co-transfection with mCherry-CHMP2A n = 12, co- transfection with mCherry-CHMP4B n = 9. ( e ) VPS4 is recruited to the centrosome independent of ESCRT III. NIH3T3 cells were co-transfected with GFP-VPS4 EQ and mCherry-CHMP6-N peptide (composed of the first 52 amino acids of CHMP6) (upper panel) or with siCHMP4B and GFP-VPS4 EQ (bottom panel). Cells were then fixed, immunostained with γ-tubulin antibodies and imaged. Left to right: entire cell (scale, 10 μm), zoomed-in images (white box) of: γ-tubulin (blue), VPS4 EQ (green) and an overlay (scale, 1 μm). White arrows mark the centrosome. CHMP6-N n = 21, siCHMP4B n = 13.

    Article Snippet: Cells were then permeabilized, blocked and immunostained with the following primary antibodies as specified in text: mouse monoclonal anti α-tubulin antibodies (1:1000, DM1A clone Sigma-Aldrich Cat # T9026), mouse monoclonal anti acetylated tubulin antibodies (1:500 or 1:1000, Thermo Scientific Cat # 32–2700 or 1:1000, Sigma-Aldrich Cat # T7451), mouse monoclonal anti γ-tubulin (1:1000, Sigma-Aldrich Cat #T6557), mouse monoclonal anti NEDD1 (1:100, Abcam Cat # ab57336), rabbit polyclonal anti γ-tubulin antibodies (1:200, Abcam Cat # ab 84355), rabbit polyclonal anti CHMP2A (1:50, Proteintech Group Cat # 10477-1-AP) rabbit polyclonal anti CHMP2B (1:50, Proteintech Group Cat # 12527-1-AP), rabbit polyclonal anti IST1 (1:50, Proteintech Group Cat # 51002-1-AP), rabbit polyclonal anti CP110 (1:50, Proteintech Group Cat # 12780-1-AP) antibodies, rabbit polyclonal anti CHMP4A (1:50, Santa Cruz Biotechnology Cat # SC-67229), rabbit polyclonal anti PCM1 antibodies (1:100, Santa Cruz Biotechnology, Cat # SC-67204), rabbit polyclonal anti Cep164 (1: 200, Abcam Cat # ab106372 or 1:200 Proteintech Group Cat # 22227-1-AP), rabbit polyclonal anti Cep290 (1:100, Abcam Cat # ab84870), mouse anti Centrin (1:500, Millipore clone 20H5, Cat # 04-1624) and rabbit polyclonal anti VTA1 antibodies (1:50, Pierce Biotechnology Cat # PA521831).

    Techniques: Transfection, Expressing, Microscopy, Cotransfection

    Ciliogenesis is defective in cells with altered VPS4 activity. ( a ) NIH3T3 transfected with the indicated plasmids or siRNA constructs were fixed, immunostained with anti-acetylated antibodies and imaged. The percentage of ciliated cells is displayed in the graph (short cilia ≤ 2 µm, cilia ≥ 2 µm). Statistical analysis was calculated using a one-way ANOVA (***p- value ≤ 0.0001). Maximum intensity projection images of representative cells are shown in the right panel. Arrows indicate cilia (scale, 10 μm). scRNA n = 113, siVPS4A/B n = 103, siCHMP2A n = 64, siCHMP2B n = 173, siCHMP4B n = 68, GFP-VPS4 EQΔMIT n = 185. Data was obtained from at least two independent experiments. GFP and GFP-VPS4 EQ transfections were repeated in each experiment for reference. For these conditions data was obtained from more than 10 experiments, n ≥ 700. ( b ) Zebrafish embryos were injected with mRNA encoding either GFP or GFP-VPS4 EQ . 32 h post injection embryos were analyzed for survival (GFP n = 112, GFP-VPS4 EQ n = 132). Live embryos were analyzed for developmental defects (GFP n = 71, GFP-VPS4 EQ n = 21). Representative images are shown. Scale, 100 μm. ( c ) Embryos (15 h) were fixed and immunostained with acetylated-tubulin antibodies. The number of cilia in the Kupffer’s vesicle of each animal was counted and embryos were categorized as control (not injected or injected with GFP), GFP-VPS4 EQ injected developmentally normal, or GFP-VPS4 EQ injected with developmental defects. Control n = 41, GFP-VPS4 EQ normal n = 17, GFP-VPS4 EQ defective n = 31. Statistical analysis was calculated using t-test. *p- value ≤ 0.1, **p- value ≤ 0.05. Scale, 10 μm. ( d ). Cells were then treated and serially sectioned as described in material and methods and imaged by TEM. Left panel: representative image of a GFP expressing cells. Second to fourth panels: representative images of cells expressing GFP-VPS4 EQ . GFP n = 16, GFP-VPS4 EQ n = 19. Scale, 0.2 μm. Right panel: percentage of ciliated cells and cells with a docked ciliary vesicle. ( e ) NIH3T3 cells, expressing PACT-TagBFP (blue) and GFP-VPS4 EQ (green) and immunostained for the cilia marker Arl13b (red) were imaged using Airyscan confocal microscopy. Scale, 0.5 μm. ( f ) NIH cells transfected with mCherry-VPS4 EQ (green) and PACT tagBFP (blue) and Smo GFP (green) were imaged live using Airyscan microscopy. Scale, 0.5 μm.

    Journal: Scientific Reports

    Article Title: VPS4 is a dynamic component of the centrosome that regulates centrosome localization of γ-tubulin, centriolar satellite stability and ciliogenesis

    doi: 10.1038/s41598-018-21491-x

    Figure Lengend Snippet: Ciliogenesis is defective in cells with altered VPS4 activity. ( a ) NIH3T3 transfected with the indicated plasmids or siRNA constructs were fixed, immunostained with anti-acetylated antibodies and imaged. The percentage of ciliated cells is displayed in the graph (short cilia ≤ 2 µm, cilia ≥ 2 µm). Statistical analysis was calculated using a one-way ANOVA (***p- value ≤ 0.0001). Maximum intensity projection images of representative cells are shown in the right panel. Arrows indicate cilia (scale, 10 μm). scRNA n = 113, siVPS4A/B n = 103, siCHMP2A n = 64, siCHMP2B n = 173, siCHMP4B n = 68, GFP-VPS4 EQΔMIT n = 185. Data was obtained from at least two independent experiments. GFP and GFP-VPS4 EQ transfections were repeated in each experiment for reference. For these conditions data was obtained from more than 10 experiments, n ≥ 700. ( b ) Zebrafish embryos were injected with mRNA encoding either GFP or GFP-VPS4 EQ . 32 h post injection embryos were analyzed for survival (GFP n = 112, GFP-VPS4 EQ n = 132). Live embryos were analyzed for developmental defects (GFP n = 71, GFP-VPS4 EQ n = 21). Representative images are shown. Scale, 100 μm. ( c ) Embryos (15 h) were fixed and immunostained with acetylated-tubulin antibodies. The number of cilia in the Kupffer’s vesicle of each animal was counted and embryos were categorized as control (not injected or injected with GFP), GFP-VPS4 EQ injected developmentally normal, or GFP-VPS4 EQ injected with developmental defects. Control n = 41, GFP-VPS4 EQ normal n = 17, GFP-VPS4 EQ defective n = 31. Statistical analysis was calculated using t-test. *p- value ≤ 0.1, **p- value ≤ 0.05. Scale, 10 μm. ( d ). Cells were then treated and serially sectioned as described in material and methods and imaged by TEM. Left panel: representative image of a GFP expressing cells. Second to fourth panels: representative images of cells expressing GFP-VPS4 EQ . GFP n = 16, GFP-VPS4 EQ n = 19. Scale, 0.2 μm. Right panel: percentage of ciliated cells and cells with a docked ciliary vesicle. ( e ) NIH3T3 cells, expressing PACT-TagBFP (blue) and GFP-VPS4 EQ (green) and immunostained for the cilia marker Arl13b (red) were imaged using Airyscan confocal microscopy. Scale, 0.5 μm. ( f ) NIH cells transfected with mCherry-VPS4 EQ (green) and PACT tagBFP (blue) and Smo GFP (green) were imaged live using Airyscan microscopy. Scale, 0.5 μm.

    Article Snippet: Cells were then permeabilized, blocked and immunostained with the following primary antibodies as specified in text: mouse monoclonal anti α-tubulin antibodies (1:1000, DM1A clone Sigma-Aldrich Cat # T9026), mouse monoclonal anti acetylated tubulin antibodies (1:500 or 1:1000, Thermo Scientific Cat # 32–2700 or 1:1000, Sigma-Aldrich Cat # T7451), mouse monoclonal anti γ-tubulin (1:1000, Sigma-Aldrich Cat #T6557), mouse monoclonal anti NEDD1 (1:100, Abcam Cat # ab57336), rabbit polyclonal anti γ-tubulin antibodies (1:200, Abcam Cat # ab 84355), rabbit polyclonal anti CHMP2A (1:50, Proteintech Group Cat # 10477-1-AP) rabbit polyclonal anti CHMP2B (1:50, Proteintech Group Cat # 12527-1-AP), rabbit polyclonal anti IST1 (1:50, Proteintech Group Cat # 51002-1-AP), rabbit polyclonal anti CP110 (1:50, Proteintech Group Cat # 12780-1-AP) antibodies, rabbit polyclonal anti CHMP4A (1:50, Santa Cruz Biotechnology Cat # SC-67229), rabbit polyclonal anti PCM1 antibodies (1:100, Santa Cruz Biotechnology, Cat # SC-67204), rabbit polyclonal anti Cep164 (1: 200, Abcam Cat # ab106372 or 1:200 Proteintech Group Cat # 22227-1-AP), rabbit polyclonal anti Cep290 (1:100, Abcam Cat # ab84870), mouse anti Centrin (1:500, Millipore clone 20H5, Cat # 04-1624) and rabbit polyclonal anti VTA1 antibodies (1:50, Pierce Biotechnology Cat # PA521831).

    Techniques: Activity Assay, Transfection, Construct, Injection, Transmission Electron Microscopy, Expressing, Marker, Confocal Microscopy, Microscopy

    A model for VPS4 function at centrosomes. ( a ) In normal conditions VPS4 dynamically associates with the centrosome. This dynamic VPS4 localization ensures proper γ-tubulin organization and MT growth at the centrosome where it also facilitates ciliogenesis. ( b ) Inhibition of VPS4 dynamic association with the centrosome using the ATP locked mutant VPS4 EQ , leads to reduced γ-tubulin levels and loss of γ-tubulin ring structure at the centrosome. Consequently, MT growth from centrosomes is impaired, centrosome positioning is misregulated, centriolar satellites are lost, and ciliogenesis is inhibited.

    Journal: Scientific Reports

    Article Title: VPS4 is a dynamic component of the centrosome that regulates centrosome localization of γ-tubulin, centriolar satellite stability and ciliogenesis

    doi: 10.1038/s41598-018-21491-x

    Figure Lengend Snippet: A model for VPS4 function at centrosomes. ( a ) In normal conditions VPS4 dynamically associates with the centrosome. This dynamic VPS4 localization ensures proper γ-tubulin organization and MT growth at the centrosome where it also facilitates ciliogenesis. ( b ) Inhibition of VPS4 dynamic association with the centrosome using the ATP locked mutant VPS4 EQ , leads to reduced γ-tubulin levels and loss of γ-tubulin ring structure at the centrosome. Consequently, MT growth from centrosomes is impaired, centrosome positioning is misregulated, centriolar satellites are lost, and ciliogenesis is inhibited.

    Article Snippet: Cells were then permeabilized, blocked and immunostained with the following primary antibodies as specified in text: mouse monoclonal anti α-tubulin antibodies (1:1000, DM1A clone Sigma-Aldrich Cat # T9026), mouse monoclonal anti acetylated tubulin antibodies (1:500 or 1:1000, Thermo Scientific Cat # 32–2700 or 1:1000, Sigma-Aldrich Cat # T7451), mouse monoclonal anti γ-tubulin (1:1000, Sigma-Aldrich Cat #T6557), mouse monoclonal anti NEDD1 (1:100, Abcam Cat # ab57336), rabbit polyclonal anti γ-tubulin antibodies (1:200, Abcam Cat # ab 84355), rabbit polyclonal anti CHMP2A (1:50, Proteintech Group Cat # 10477-1-AP) rabbit polyclonal anti CHMP2B (1:50, Proteintech Group Cat # 12527-1-AP), rabbit polyclonal anti IST1 (1:50, Proteintech Group Cat # 51002-1-AP), rabbit polyclonal anti CP110 (1:50, Proteintech Group Cat # 12780-1-AP) antibodies, rabbit polyclonal anti CHMP4A (1:50, Santa Cruz Biotechnology Cat # SC-67229), rabbit polyclonal anti PCM1 antibodies (1:100, Santa Cruz Biotechnology, Cat # SC-67204), rabbit polyclonal anti Cep164 (1: 200, Abcam Cat # ab106372 or 1:200 Proteintech Group Cat # 22227-1-AP), rabbit polyclonal anti Cep290 (1:100, Abcam Cat # ab84870), mouse anti Centrin (1:500, Millipore clone 20H5, Cat # 04-1624) and rabbit polyclonal anti VTA1 antibodies (1:50, Pierce Biotechnology Cat # PA521831).

    Techniques: Inhibition, Mutagenesis

    Expression of VPS4 EQ causes reduced γ-tubulin staining at centrosomes but does not affect overall centriolar structure. ( a ) NIH3T3 cells grown on gridded coverslips were fixed and imaged to locate cells expressing GFP or GFP-VPS4 EQ ). Scale, 0.2 μm. ( b–d ) The organization of known centrosomal proteins was tested in fixed NIH3T3 cells expressing GFP (control), GFP-VPS4 or GFP- VPS4 EQ , immunostained with the indicated antibodies. Cells were imaged using 3D SIM. Shown are maximum intensity projections of reconstructed images from representative cells. Each panel shows (from left to right) the entire cell (scale, 5 μm); zoomed-in images (white box) of each channel and a zoomed-in overlay image (scale, 0.2 μm). ( b ) Endogenous CP110 (antibody staining, blue) GFP n = 59, VPS4 n = 10, VPS4 EQ n = 20. ( c ) Endogenous Cep164 (antibody staining, blue) GFP n = 10, GFP-VPS4 n = 8, GFP- VPS4 EQ n = 15. ( d ) Endogenous γ-tubulin (antibody staining, blue). GFP n = 20, GFP-VPS4 n = 15, GFP-VPS4 EQ n = 21. Note that while CP110 and Cep164 are not affected by VPS4 EQ expression, γ tubulin staining is severely reduced.

    Journal: Scientific Reports

    Article Title: VPS4 is a dynamic component of the centrosome that regulates centrosome localization of γ-tubulin, centriolar satellite stability and ciliogenesis

    doi: 10.1038/s41598-018-21491-x

    Figure Lengend Snippet: Expression of VPS4 EQ causes reduced γ-tubulin staining at centrosomes but does not affect overall centriolar structure. ( a ) NIH3T3 cells grown on gridded coverslips were fixed and imaged to locate cells expressing GFP or GFP-VPS4 EQ ). Scale, 0.2 μm. ( b–d ) The organization of known centrosomal proteins was tested in fixed NIH3T3 cells expressing GFP (control), GFP-VPS4 or GFP- VPS4 EQ , immunostained with the indicated antibodies. Cells were imaged using 3D SIM. Shown are maximum intensity projections of reconstructed images from representative cells. Each panel shows (from left to right) the entire cell (scale, 5 μm); zoomed-in images (white box) of each channel and a zoomed-in overlay image (scale, 0.2 μm). ( b ) Endogenous CP110 (antibody staining, blue) GFP n = 59, VPS4 n = 10, VPS4 EQ n = 20. ( c ) Endogenous Cep164 (antibody staining, blue) GFP n = 10, GFP-VPS4 n = 8, GFP- VPS4 EQ n = 15. ( d ) Endogenous γ-tubulin (antibody staining, blue). GFP n = 20, GFP-VPS4 n = 15, GFP-VPS4 EQ n = 21. Note that while CP110 and Cep164 are not affected by VPS4 EQ expression, γ tubulin staining is severely reduced.

    Article Snippet: Cells were then permeabilized, blocked and immunostained with the following primary antibodies as specified in text: mouse monoclonal anti α-tubulin antibodies (1:1000, DM1A clone Sigma-Aldrich Cat # T9026), mouse monoclonal anti acetylated tubulin antibodies (1:500 or 1:1000, Thermo Scientific Cat # 32–2700 or 1:1000, Sigma-Aldrich Cat # T7451), mouse monoclonal anti γ-tubulin (1:1000, Sigma-Aldrich Cat #T6557), mouse monoclonal anti NEDD1 (1:100, Abcam Cat # ab57336), rabbit polyclonal anti γ-tubulin antibodies (1:200, Abcam Cat # ab 84355), rabbit polyclonal anti CHMP2A (1:50, Proteintech Group Cat # 10477-1-AP) rabbit polyclonal anti CHMP2B (1:50, Proteintech Group Cat # 12527-1-AP), rabbit polyclonal anti IST1 (1:50, Proteintech Group Cat # 51002-1-AP), rabbit polyclonal anti CP110 (1:50, Proteintech Group Cat # 12780-1-AP) antibodies, rabbit polyclonal anti CHMP4A (1:50, Santa Cruz Biotechnology Cat # SC-67229), rabbit polyclonal anti PCM1 antibodies (1:100, Santa Cruz Biotechnology, Cat # SC-67204), rabbit polyclonal anti Cep164 (1: 200, Abcam Cat # ab106372 or 1:200 Proteintech Group Cat # 22227-1-AP), rabbit polyclonal anti Cep290 (1:100, Abcam Cat # ab84870), mouse anti Centrin (1:500, Millipore clone 20H5, Cat # 04-1624) and rabbit polyclonal anti VTA1 antibodies (1:50, Pierce Biotechnology Cat # PA521831).

    Techniques: Expressing, Staining

    Radial MT growth and centrosome positioning are abnormal upon perturbation of VPS4 ATPase activity. ( a ) NIH3T3 cells were co-transfected with EB1-GFP, a MT plus-end binding protein, and with either mCherry or mCherry-VPS4 EQ ). mCherry n = 14, mCherry-VPS4 EQ n = 19. Graph on right: percentage of cells in which radial MT growth was observed. Statistical analysis for normal radial MT was calculated using t-test ***p- value ≤ 0.0001. Scale, 10 μm. ( b ) Fixed NIH3T3 cells expressing either GFP or GFP-VPS4 EQ were immunostained with anti-acetylated tubulin (red) and anti γ-tubulin (blue) antibodies and imaged in 3D using SIM. Maximum intensity projections of representative images are shown. GFP n = 147, GFP-VPS4 EQ n = 115. White arrows indicate the centrosome. Graph on right: percentage of cells exhibiting normal or heavy acetylation. Statistical analysis for normal acetylation was calculated using t-test ***p- value ≤ 0.0001. Scale, 1 μm. ( c ) NIH3T3 cells were transfected with the centrosome marker PACT-mRFP (red) and with either GFP or GFP-VPS4 EQ (green). 24 h post transfection cells were plated on fibronectin coated micropatterns (as described in materials and methods), fixed, stained with Hoechst (blue) and imaged in 3D using a spinning disk confocal microscope. Shown are maximum intensity Y projection images of representative cells. The distance between the centrosome and the center of the nucleus (see cartoon on the right) was measured in 3D for each cell as described in materials and methods and plotted in a histogram (bottom panel). GFP n = 45, GFP-VPS4 EQ n = 50. Scale, 10 μm.

    Journal: Scientific Reports

    Article Title: VPS4 is a dynamic component of the centrosome that regulates centrosome localization of γ-tubulin, centriolar satellite stability and ciliogenesis

    doi: 10.1038/s41598-018-21491-x

    Figure Lengend Snippet: Radial MT growth and centrosome positioning are abnormal upon perturbation of VPS4 ATPase activity. ( a ) NIH3T3 cells were co-transfected with EB1-GFP, a MT plus-end binding protein, and with either mCherry or mCherry-VPS4 EQ ). mCherry n = 14, mCherry-VPS4 EQ n = 19. Graph on right: percentage of cells in which radial MT growth was observed. Statistical analysis for normal radial MT was calculated using t-test ***p- value ≤ 0.0001. Scale, 10 μm. ( b ) Fixed NIH3T3 cells expressing either GFP or GFP-VPS4 EQ were immunostained with anti-acetylated tubulin (red) and anti γ-tubulin (blue) antibodies and imaged in 3D using SIM. Maximum intensity projections of representative images are shown. GFP n = 147, GFP-VPS4 EQ n = 115. White arrows indicate the centrosome. Graph on right: percentage of cells exhibiting normal or heavy acetylation. Statistical analysis for normal acetylation was calculated using t-test ***p- value ≤ 0.0001. Scale, 1 μm. ( c ) NIH3T3 cells were transfected with the centrosome marker PACT-mRFP (red) and with either GFP or GFP-VPS4 EQ (green). 24 h post transfection cells were plated on fibronectin coated micropatterns (as described in materials and methods), fixed, stained with Hoechst (blue) and imaged in 3D using a spinning disk confocal microscope. Shown are maximum intensity Y projection images of representative cells. The distance between the centrosome and the center of the nucleus (see cartoon on the right) was measured in 3D for each cell as described in materials and methods and plotted in a histogram (bottom panel). GFP n = 45, GFP-VPS4 EQ n = 50. Scale, 10 μm.

    Article Snippet: Cells were then permeabilized, blocked and immunostained with the following primary antibodies as specified in text: mouse monoclonal anti α-tubulin antibodies (1:1000, DM1A clone Sigma-Aldrich Cat # T9026), mouse monoclonal anti acetylated tubulin antibodies (1:500 or 1:1000, Thermo Scientific Cat # 32–2700 or 1:1000, Sigma-Aldrich Cat # T7451), mouse monoclonal anti γ-tubulin (1:1000, Sigma-Aldrich Cat #T6557), mouse monoclonal anti NEDD1 (1:100, Abcam Cat # ab57336), rabbit polyclonal anti γ-tubulin antibodies (1:200, Abcam Cat # ab 84355), rabbit polyclonal anti CHMP2A (1:50, Proteintech Group Cat # 10477-1-AP) rabbit polyclonal anti CHMP2B (1:50, Proteintech Group Cat # 12527-1-AP), rabbit polyclonal anti IST1 (1:50, Proteintech Group Cat # 51002-1-AP), rabbit polyclonal anti CP110 (1:50, Proteintech Group Cat # 12780-1-AP) antibodies, rabbit polyclonal anti CHMP4A (1:50, Santa Cruz Biotechnology Cat # SC-67229), rabbit polyclonal anti PCM1 antibodies (1:100, Santa Cruz Biotechnology, Cat # SC-67204), rabbit polyclonal anti Cep164 (1: 200, Abcam Cat # ab106372 or 1:200 Proteintech Group Cat # 22227-1-AP), rabbit polyclonal anti Cep290 (1:100, Abcam Cat # ab84870), mouse anti Centrin (1:500, Millipore clone 20H5, Cat # 04-1624) and rabbit polyclonal anti VTA1 antibodies (1:50, Pierce Biotechnology Cat # PA521831).

    Techniques: Activity Assay, Transfection, Binding Assay, Expressing, Marker, Staining, Microscopy

    Localization and dynamics of VPS4 at the centrosomes of interphase cells. ( a ) NIH3T3 cells were transfected with the centrosome marker, PACT-mRFP, and GFP-VPS4 or GFP-VPS4 EQ and imaged by Airyscan confocal microscopy. Images were processed to reveal the sub-diffraction localization of the proteins. Both the wild-type and the ATPase deficient VPS4 localize to the centrosome. Left to right: entire cell (scale, 5 μm), zoomed-in images (white box) of: PACT (red), VPS4 (green), an overlay and a YZ projection (scale, 0.5 μm). ( b ) Top panel: NIH3T3 cells transfected with GFP, GFP-VPS4 or GFP-VPS4 EQ ). GFP-VPS4 and GFP-VPS4 EQ express at similar levels. Bottom panel: Cells expressing GFP, GFP-VPS4, or GFP-VPS4 EQ together with PACT-TagBFP were fixed and stained with anti-centrin. Z stacks of centrosomes were collected based on centrosomal markers using Airyscan microscopy. GFP intensity at the centrosome was quantified as described in materials and methods. Shown are average intensity values that were normalized to average GFP intensity obtained in control cells. GFP n = 56, GFP-VPS4 n = 35, GFP-VPS4 EQ n = 52 from 2 independent experiments. ( c ) Cells transfected with PACT-mRFP and GFP-VPS4 or GFP-VPS4 EQ were grown in high glucose media, fixed, and stained with Hoecst and antibodies to acetylated tubulin. GFP-VPS4 EQ expressing cells in anaphase or metaphase like the one shown here were rare. Scale, whole cells, 5 μm; zoomed-in image, 1 μm. ( d ) iFRAP recordings of NIH3T3 cells expressing PACT-mRFP and either GFP-VPS4 or GFP-VPS4 EQ were performed using Airyscan Confocal imaging in Fast mode. A single plane was imaged and then two regions on either side of the centrosome (covering most of the cell) were photobleached (white frame box). Image capture alternated with photobleaching for 90–100 time points. The intensity of the GFP signal at the centrosome was quantified using the location of the PACT-mRFP marker to generate the analysis region (small yellow frame box). Intensity was normalized to the pre-bleach intensity and the mean+/− the standard deviation was plotted for GFP-VPS4 and GFP-VPS4 EQ (top right panel). Bottom panel: zoomed-in images taken from yellow frame boxes in the corresponding upper panels are shown. GFP-VPS4 n = 11, GFP-VPS4 EQ n = 5. Scale, upper panel, 5 μm; bottom panel, 0.5 μm.

    Journal: Scientific Reports

    Article Title: VPS4 is a dynamic component of the centrosome that regulates centrosome localization of γ-tubulin, centriolar satellite stability and ciliogenesis

    doi: 10.1038/s41598-018-21491-x

    Figure Lengend Snippet: Localization and dynamics of VPS4 at the centrosomes of interphase cells. ( a ) NIH3T3 cells were transfected with the centrosome marker, PACT-mRFP, and GFP-VPS4 or GFP-VPS4 EQ and imaged by Airyscan confocal microscopy. Images were processed to reveal the sub-diffraction localization of the proteins. Both the wild-type and the ATPase deficient VPS4 localize to the centrosome. Left to right: entire cell (scale, 5 μm), zoomed-in images (white box) of: PACT (red), VPS4 (green), an overlay and a YZ projection (scale, 0.5 μm). ( b ) Top panel: NIH3T3 cells transfected with GFP, GFP-VPS4 or GFP-VPS4 EQ ). GFP-VPS4 and GFP-VPS4 EQ express at similar levels. Bottom panel: Cells expressing GFP, GFP-VPS4, or GFP-VPS4 EQ together with PACT-TagBFP were fixed and stained with anti-centrin. Z stacks of centrosomes were collected based on centrosomal markers using Airyscan microscopy. GFP intensity at the centrosome was quantified as described in materials and methods. Shown are average intensity values that were normalized to average GFP intensity obtained in control cells. GFP n = 56, GFP-VPS4 n = 35, GFP-VPS4 EQ n = 52 from 2 independent experiments. ( c ) Cells transfected with PACT-mRFP and GFP-VPS4 or GFP-VPS4 EQ were grown in high glucose media, fixed, and stained with Hoecst and antibodies to acetylated tubulin. GFP-VPS4 EQ expressing cells in anaphase or metaphase like the one shown here were rare. Scale, whole cells, 5 μm; zoomed-in image, 1 μm. ( d ) iFRAP recordings of NIH3T3 cells expressing PACT-mRFP and either GFP-VPS4 or GFP-VPS4 EQ were performed using Airyscan Confocal imaging in Fast mode. A single plane was imaged and then two regions on either side of the centrosome (covering most of the cell) were photobleached (white frame box). Image capture alternated with photobleaching for 90–100 time points. The intensity of the GFP signal at the centrosome was quantified using the location of the PACT-mRFP marker to generate the analysis region (small yellow frame box). Intensity was normalized to the pre-bleach intensity and the mean+/− the standard deviation was plotted for GFP-VPS4 and GFP-VPS4 EQ (top right panel). Bottom panel: zoomed-in images taken from yellow frame boxes in the corresponding upper panels are shown. GFP-VPS4 n = 11, GFP-VPS4 EQ n = 5. Scale, upper panel, 5 μm; bottom panel, 0.5 μm.

    Article Snippet: Cells were then permeabilized, blocked and immunostained with the following primary antibodies as specified in text: mouse monoclonal anti α-tubulin antibodies (1:1000, DM1A clone Sigma-Aldrich Cat # T9026), mouse monoclonal anti acetylated tubulin antibodies (1:500 or 1:1000, Thermo Scientific Cat # 32–2700 or 1:1000, Sigma-Aldrich Cat # T7451), mouse monoclonal anti γ-tubulin (1:1000, Sigma-Aldrich Cat #T6557), mouse monoclonal anti NEDD1 (1:100, Abcam Cat # ab57336), rabbit polyclonal anti γ-tubulin antibodies (1:200, Abcam Cat # ab 84355), rabbit polyclonal anti CHMP2A (1:50, Proteintech Group Cat # 10477-1-AP) rabbit polyclonal anti CHMP2B (1:50, Proteintech Group Cat # 12527-1-AP), rabbit polyclonal anti IST1 (1:50, Proteintech Group Cat # 51002-1-AP), rabbit polyclonal anti CP110 (1:50, Proteintech Group Cat # 12780-1-AP) antibodies, rabbit polyclonal anti CHMP4A (1:50, Santa Cruz Biotechnology Cat # SC-67229), rabbit polyclonal anti PCM1 antibodies (1:100, Santa Cruz Biotechnology, Cat # SC-67204), rabbit polyclonal anti Cep164 (1: 200, Abcam Cat # ab106372 or 1:200 Proteintech Group Cat # 22227-1-AP), rabbit polyclonal anti Cep290 (1:100, Abcam Cat # ab84870), mouse anti Centrin (1:500, Millipore clone 20H5, Cat # 04-1624) and rabbit polyclonal anti VTA1 antibodies (1:50, Pierce Biotechnology Cat # PA521831).

    Techniques: Transfection, Marker, Confocal Microscopy, Expressing, Staining, Microscopy, Imaging, Standard Deviation

    Representative western blotting of intercalated disc proteins and β-tubulin in the right ventricular myocardium from patients with AC and healthy controls. (A) Western blotting of myocardial intercalated disc proteins in the controls, and AC patients with and without DSG2 variation, along with the quantification of the protein expression; (B) western blotting of myocardial intercalated disc proteins in the controls, and AC patients with and without PKP2 variation, along with the quantification of the protein expression. DSG2, desmoglein 2; JUP, junction plakoglobin; Cx43, connexin 43; PKP2, plakophilin 2.

    Journal: Experimental and Therapeutic Medicine

    Article Title: Arrhythmogenic cardiomyopathy: Identification of desmosomal gene variations and desmosomal protein expression in variation carriers

    doi: 10.3892/etm.2018.5694

    Figure Lengend Snippet: Representative western blotting of intercalated disc proteins and β-tubulin in the right ventricular myocardium from patients with AC and healthy controls. (A) Western blotting of myocardial intercalated disc proteins in the controls, and AC patients with and without DSG2 variation, along with the quantification of the protein expression; (B) western blotting of myocardial intercalated disc proteins in the controls, and AC patients with and without PKP2 variation, along with the quantification of the protein expression. DSG2, desmoglein 2; JUP, junction plakoglobin; Cx43, connexin 43; PKP2, plakophilin 2.

    Article Snippet: Assessment of the cytoskeletal proteins was performed using the following primary antibodies: Rabbit anti-DSG2 (ab85632; 1:1,000), anti-JUP (2309; 1:50), anti-Cx43 (3512; 1:100) and mouse anti-PKP2 (ab151402; 1:200), and the loading control was β-tubulin (2146; 1:1,000; Cell Signaling Technology, Inc.) was used as the internal reference.

    Techniques: Western Blot, Expressing

    The KIF17 CLS is necessary and sufficient for ciliary localization. ( a ) Odora, MDCK II, NIH3T3, and hTERT-RPE cells expressing full length KIF17-mCit (green) were fixed and stained for acetylated tubulin to mark cilia (red). Top row, images of entire cells; bottom row, higher magnification of cilia in boxed areas. White arrowheads indicate distal tips of cilia. ( b ) Left, schematic of full length human KIF17. NC, neck coil; CC, coiled-coil. Right, NIH3T3 cells expressing KIF17-mCit (green) were fixed and stained for acetylated tubulin (red) to mark cilia and γ-tubulin (blue) to mark the basal body. ( c-g ) Schematics of truncated and mutant KIF17 constructs (left) and their localization in Odora cells (right). Cells expressing the indicated truncated or mutant KIF17 motors (green) were fixed and stained for acetylated tubulin (red in c,f,g) or the myc tag (white in d). ( h ) Odora cells expressing full length KHC-mCit or KHC fused with the wildtype or mutant versions of the KIF17 tail were fixed and stained with antibodies to acetylated tubulin (red). Scale bars throughout figure are either 10 μm for images of entire cell or 1 μm for cilia.

    Journal: Nature cell biology

    Article Title: Ciliary entry of the kinesin-2 motor KIF17 is regulated by importin-?2 and Ran-GTP

    doi: 10.1038/ncb2073

    Figure Lengend Snippet: The KIF17 CLS is necessary and sufficient for ciliary localization. ( a ) Odora, MDCK II, NIH3T3, and hTERT-RPE cells expressing full length KIF17-mCit (green) were fixed and stained for acetylated tubulin to mark cilia (red). Top row, images of entire cells; bottom row, higher magnification of cilia in boxed areas. White arrowheads indicate distal tips of cilia. ( b ) Left, schematic of full length human KIF17. NC, neck coil; CC, coiled-coil. Right, NIH3T3 cells expressing KIF17-mCit (green) were fixed and stained for acetylated tubulin (red) to mark cilia and γ-tubulin (blue) to mark the basal body. ( c-g ) Schematics of truncated and mutant KIF17 constructs (left) and their localization in Odora cells (right). Cells expressing the indicated truncated or mutant KIF17 motors (green) were fixed and stained for acetylated tubulin (red in c,f,g) or the myc tag (white in d). ( h ) Odora cells expressing full length KHC-mCit or KHC fused with the wildtype or mutant versions of the KIF17 tail were fixed and stained with antibodies to acetylated tubulin (red). Scale bars throughout figure are either 10 μm for images of entire cell or 1 μm for cilia.

    Article Snippet: Antibodies and plasmids Commercial antibodies include: γ-tubulin (polyclonal and GTU-88 clone, Sigma), myc (9E10 clone, Sigma), importin-β2 (558660, BD Pharmingen), importin-β1 (610559, BD Transduction Laboratories), FLAG (F7425 or M2 clone, Sigma), Ran (610340, BD Transduction Laboratories), and adenylyl cyclase III (Santa Cruz Biotechnology).

    Techniques: Expressing, Staining, Mutagenesis, Construct