rabbit anti gfp  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit anti gfp
    (A) Ccdc66 localizes to the axoneme of the primary cilium. Endogenous staining of Ccdc66 in ciliated IMCD3 cells fixed with 4% PFA was performed using homemade antibody raised against the mouse Ccdc66 protein. Cells were co-stained <t>with</t> <t>anti-acetylated-tubulin,</t> anti-Arl13b and DAPI. The top panel shows images obtained using structured illumination microscopy (SIM). The bottom panel shows confocal microscopy images of samples processed by ultra-structure expansion microscopy (U-ExM). Scale bars SIM: 1 µm, Scale bars U-ExM: 222 nm (B-D) Effects of CCDC66 depletion on cilia formation and length. IMCD3 cells transduced and stably expressing either control or shRNA targeting C-terminus of Ccdc66 (shCcdc66) were grown on glass coverslips, fixed with 4% PFA after 48 h of serum starvation and imaged with confocal microscopy. Scale bar: 5 µm. Insets show 3× magnifications of the cilia, Scale bar: 2 µm (C) Quantification of the percentage of cells with cilia (B). Data represents the mean ± SD of 2 independent experiments. n > 600 cells for control and >400 cells for Ccdc66 depletion. The mean cilia percentage is 84.67% for shControl and 78.72% for shCcdc66. (Welch’s t test, ns: not significant) (D) Quantification of cilia length in 3D, according to acetylated-tubulin signal (co-localizing with Arl13b) (B). The super plot represents the mean ± SEM of 2 independent experiments superimposed onto a scatter plot of normalized individual experimental values to control. The first experimental replicate is shown as a darker circle, and the second as a lighter-colored square. Individual values are represented in lighter shades than the corresponding averages. n > 200 cells for each condition. The mean cilia length in Ccdc66-depleted cells decreased to 0.55-fold compared to the mean control length. (Unpaired t test, *p=0.0114) (E-G) Ccdc66 depletion leads to frequent length fluctuations and enhanced ectocytosis in steady-state cilia. (E) The <t>IMCD3::SSTR3-GFP</t> cells transduced with either control or Ccdc66 shRNA virus were grown in FluoroDish and serum starved for 48 h, then imaged with confocal microscopy with 63× objective. Images were acquired every 4 min for 6 h. Still images are inverted to emphasize cilia better. Scale bar: 3 μm. (F) Ciliary kinetics are presented as normalized length curves, measured from SSTR3-GFP fluorescence in two independent experiments, represented as the mean ± SD. n=20 cilia for both shControl and shCcdc66 conditions. (G) Quantification of ciliary ectocytosis events in (E). Ciliary vesicle or fragment ectocytosis were categorized based on the size of the released EVs, vesicle <500 nm and fragment >500 nm, and plotted as a bar plot. (p values of Welch’s t test, *p=0.0497, **p=0.0095, ns=not significant) Ccdc66, coiled-coil domain-containing protein 66; IMCD3, inner medullary collecting duct cell line; PFA, paraformaldehyde; Arl13b, ADP-ribosylation factor-like protein 13B; DAPI, 4′,6-diamidino-2-phenylindole; shRNA, short hairpin RNA; SSTR3, Somatostatin receptor type 3; GFP, green fluorescent protein; EV, extracellular vesicle; MT, microtubules; DMSO, dimethyl sulfoxide; SEM, standard error of mean; SD, standard deviation.
    Rabbit Anti Gfp, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti gfp/product/Cell Signaling Technology Inc
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti gfp - by Bioz Stars, 2024-06
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    Images

    1) Product Images from "Ccdc66 regulates primary cilium stability, disassembly and signaling important for epithelial organization"

    Article Title: Ccdc66 regulates primary cilium stability, disassembly and signaling important for epithelial organization

    Journal: bioRxiv

    doi: 10.1101/2024.06.16.599243

    (A) Ccdc66 localizes to the axoneme of the primary cilium. Endogenous staining of Ccdc66 in ciliated IMCD3 cells fixed with 4% PFA was performed using homemade antibody raised against the mouse Ccdc66 protein. Cells were co-stained with anti-acetylated-tubulin, anti-Arl13b and DAPI. The top panel shows images obtained using structured illumination microscopy (SIM). The bottom panel shows confocal microscopy images of samples processed by ultra-structure expansion microscopy (U-ExM). Scale bars SIM: 1 µm, Scale bars U-ExM: 222 nm (B-D) Effects of CCDC66 depletion on cilia formation and length. IMCD3 cells transduced and stably expressing either control or shRNA targeting C-terminus of Ccdc66 (shCcdc66) were grown on glass coverslips, fixed with 4% PFA after 48 h of serum starvation and imaged with confocal microscopy. Scale bar: 5 µm. Insets show 3× magnifications of the cilia, Scale bar: 2 µm (C) Quantification of the percentage of cells with cilia (B). Data represents the mean ± SD of 2 independent experiments. n > 600 cells for control and >400 cells for Ccdc66 depletion. The mean cilia percentage is 84.67% for shControl and 78.72% for shCcdc66. (Welch’s t test, ns: not significant) (D) Quantification of cilia length in 3D, according to acetylated-tubulin signal (co-localizing with Arl13b) (B). The super plot represents the mean ± SEM of 2 independent experiments superimposed onto a scatter plot of normalized individual experimental values to control. The first experimental replicate is shown as a darker circle, and the second as a lighter-colored square. Individual values are represented in lighter shades than the corresponding averages. n > 200 cells for each condition. The mean cilia length in Ccdc66-depleted cells decreased to 0.55-fold compared to the mean control length. (Unpaired t test, *p=0.0114) (E-G) Ccdc66 depletion leads to frequent length fluctuations and enhanced ectocytosis in steady-state cilia. (E) The IMCD3::SSTR3-GFP cells transduced with either control or Ccdc66 shRNA virus were grown in FluoroDish and serum starved for 48 h, then imaged with confocal microscopy with 63× objective. Images were acquired every 4 min for 6 h. Still images are inverted to emphasize cilia better. Scale bar: 3 μm. (F) Ciliary kinetics are presented as normalized length curves, measured from SSTR3-GFP fluorescence in two independent experiments, represented as the mean ± SD. n=20 cilia for both shControl and shCcdc66 conditions. (G) Quantification of ciliary ectocytosis events in (E). Ciliary vesicle or fragment ectocytosis were categorized based on the size of the released EVs, vesicle <500 nm and fragment >500 nm, and plotted as a bar plot. (p values of Welch’s t test, *p=0.0497, **p=0.0095, ns=not significant) Ccdc66, coiled-coil domain-containing protein 66; IMCD3, inner medullary collecting duct cell line; PFA, paraformaldehyde; Arl13b, ADP-ribosylation factor-like protein 13B; DAPI, 4′,6-diamidino-2-phenylindole; shRNA, short hairpin RNA; SSTR3, Somatostatin receptor type 3; GFP, green fluorescent protein; EV, extracellular vesicle; MT, microtubules; DMSO, dimethyl sulfoxide; SEM, standard error of mean; SD, standard deviation.
    Figure Legend Snippet: (A) Ccdc66 localizes to the axoneme of the primary cilium. Endogenous staining of Ccdc66 in ciliated IMCD3 cells fixed with 4% PFA was performed using homemade antibody raised against the mouse Ccdc66 protein. Cells were co-stained with anti-acetylated-tubulin, anti-Arl13b and DAPI. The top panel shows images obtained using structured illumination microscopy (SIM). The bottom panel shows confocal microscopy images of samples processed by ultra-structure expansion microscopy (U-ExM). Scale bars SIM: 1 µm, Scale bars U-ExM: 222 nm (B-D) Effects of CCDC66 depletion on cilia formation and length. IMCD3 cells transduced and stably expressing either control or shRNA targeting C-terminus of Ccdc66 (shCcdc66) were grown on glass coverslips, fixed with 4% PFA after 48 h of serum starvation and imaged with confocal microscopy. Scale bar: 5 µm. Insets show 3× magnifications of the cilia, Scale bar: 2 µm (C) Quantification of the percentage of cells with cilia (B). Data represents the mean ± SD of 2 independent experiments. n > 600 cells for control and >400 cells for Ccdc66 depletion. The mean cilia percentage is 84.67% for shControl and 78.72% for shCcdc66. (Welch’s t test, ns: not significant) (D) Quantification of cilia length in 3D, according to acetylated-tubulin signal (co-localizing with Arl13b) (B). The super plot represents the mean ± SEM of 2 independent experiments superimposed onto a scatter plot of normalized individual experimental values to control. The first experimental replicate is shown as a darker circle, and the second as a lighter-colored square. Individual values are represented in lighter shades than the corresponding averages. n > 200 cells for each condition. The mean cilia length in Ccdc66-depleted cells decreased to 0.55-fold compared to the mean control length. (Unpaired t test, *p=0.0114) (E-G) Ccdc66 depletion leads to frequent length fluctuations and enhanced ectocytosis in steady-state cilia. (E) The IMCD3::SSTR3-GFP cells transduced with either control or Ccdc66 shRNA virus were grown in FluoroDish and serum starved for 48 h, then imaged with confocal microscopy with 63× objective. Images were acquired every 4 min for 6 h. Still images are inverted to emphasize cilia better. Scale bar: 3 μm. (F) Ciliary kinetics are presented as normalized length curves, measured from SSTR3-GFP fluorescence in two independent experiments, represented as the mean ± SD. n=20 cilia for both shControl and shCcdc66 conditions. (G) Quantification of ciliary ectocytosis events in (E). Ciliary vesicle or fragment ectocytosis were categorized based on the size of the released EVs, vesicle <500 nm and fragment >500 nm, and plotted as a bar plot. (p values of Welch’s t test, *p=0.0497, **p=0.0095, ns=not significant) Ccdc66, coiled-coil domain-containing protein 66; IMCD3, inner medullary collecting duct cell line; PFA, paraformaldehyde; Arl13b, ADP-ribosylation factor-like protein 13B; DAPI, 4′,6-diamidino-2-phenylindole; shRNA, short hairpin RNA; SSTR3, Somatostatin receptor type 3; GFP, green fluorescent protein; EV, extracellular vesicle; MT, microtubules; DMSO, dimethyl sulfoxide; SEM, standard error of mean; SD, standard deviation.

    Techniques Used: Staining, Microscopy, Confocal Microscopy, Stable Transfection, Expressing, Control, shRNA, Transduction, Virus, Fluorescence, Standard Deviation

    Live imaging of Ccdc66-depleted cells reveals an increase in ectocytosis and frequent fluctuations in cilia length. (A-C) Cilium disassembly kinetics upon serum stimulation. The IMCD3::SSTR3-GFP cells transduced with either control or Ccdc66 shRNA were grown and serum starved in FluoroDish for 48 h, then imaged with confocal microscopy for 6 h immediately upon serum addition. With images acquired every 4 min. Representative images show one control cilium that gradually resorbs, and three Ccdc66-depleted cilia that undergo disassembly through gradual resorption, instant loss (whole cilium shedding), or a combination of both. Still images are inverted to emphasize cilia better. Scale bar 3 μm (B) Normalized cilia length curves are measured from fluorescence of two independent experiments and represented as the mean ± SD. n=20 cilia for both shControl and shCcdc66 conditions. (C) Quantification of ciliary ectocytosis events in (A). Ciliary events, including vesicle releases (<500 nm) and cilium fragment shedding (>500 nm), were categorized based on the size of the released extracellular vesicles (EVs) and plotted as a bar plot (p values of Welch’s t test *p=0.0272, *p=0.0240, *p=0.0186). (D) Media from IMCD3::SSTR3-GFP was collected 6 h post serum stimulation and separated by high speed centrifugation for subsequent western blot. Pelleted material shows the presence of ciliary markers at expected molecular weights suggesting pelleting of ciliary EVs and fragments. Membranes are blotted with anti-GFP and anti-acetylated tubulin. Shown in the graph is the fold change in the intensity of the acetylated tubulin bands, normalized to shControl of the displayed experimental replicate. The sample was normalized to total cell protein abundance before loading to account for differences in cell numbers between control and Ccdc66-depleted cells. n=2. The bar plot represents mean ± SD of 2 independent experiments. (One sample t test p value **p=0.0087) Ccdc66, coiled-coil domain-containing protein 66; IMCD3, inner medullary collecting duct cell line; SSTR3, Somatostatin receptor type 3; GFP, green fluorescent protein; shRNA, short hairpin RNA; SD, standard deviation.
    Figure Legend Snippet: Live imaging of Ccdc66-depleted cells reveals an increase in ectocytosis and frequent fluctuations in cilia length. (A-C) Cilium disassembly kinetics upon serum stimulation. The IMCD3::SSTR3-GFP cells transduced with either control or Ccdc66 shRNA were grown and serum starved in FluoroDish for 48 h, then imaged with confocal microscopy for 6 h immediately upon serum addition. With images acquired every 4 min. Representative images show one control cilium that gradually resorbs, and three Ccdc66-depleted cilia that undergo disassembly through gradual resorption, instant loss (whole cilium shedding), or a combination of both. Still images are inverted to emphasize cilia better. Scale bar 3 μm (B) Normalized cilia length curves are measured from fluorescence of two independent experiments and represented as the mean ± SD. n=20 cilia for both shControl and shCcdc66 conditions. (C) Quantification of ciliary ectocytosis events in (A). Ciliary events, including vesicle releases (<500 nm) and cilium fragment shedding (>500 nm), were categorized based on the size of the released extracellular vesicles (EVs) and plotted as a bar plot (p values of Welch’s t test *p=0.0272, *p=0.0240, *p=0.0186). (D) Media from IMCD3::SSTR3-GFP was collected 6 h post serum stimulation and separated by high speed centrifugation for subsequent western blot. Pelleted material shows the presence of ciliary markers at expected molecular weights suggesting pelleting of ciliary EVs and fragments. Membranes are blotted with anti-GFP and anti-acetylated tubulin. Shown in the graph is the fold change in the intensity of the acetylated tubulin bands, normalized to shControl of the displayed experimental replicate. The sample was normalized to total cell protein abundance before loading to account for differences in cell numbers between control and Ccdc66-depleted cells. n=2. The bar plot represents mean ± SD of 2 independent experiments. (One sample t test p value **p=0.0087) Ccdc66, coiled-coil domain-containing protein 66; IMCD3, inner medullary collecting duct cell line; SSTR3, Somatostatin receptor type 3; GFP, green fluorescent protein; shRNA, short hairpin RNA; SD, standard deviation.

    Techniques Used: Imaging, Transduction, Control, shRNA, Confocal Microscopy, Fluorescence, Centrifugation, Western Blot, Standard Deviation

    gfp  (Cell Signaling Technology Inc)


    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
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    Cell Signaling Technology Inc gfp

    Gfp, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gfp/product/Cell Signaling Technology Inc
    Average 86 stars, based on 1 article reviews
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    gfp - by Bioz Stars, 2024-06
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    Images

    1) Product Images from "Mitoferrin2 is a synthetic lethal target for chromosome 8p deleted cancers"

    Article Title: Mitoferrin2 is a synthetic lethal target for chromosome 8p deleted cancers

    Journal: Genome Medicine

    doi: 10.1186/s13073-024-01357-w


    Figure Legend Snippet:

    Techniques Used: Western Blot

    rabbit monoclonal anti gfp  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit monoclonal anti gfp

    Rabbit Monoclonal Anti Gfp, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit monoclonal anti gfp/product/Cell Signaling Technology Inc
    Average 86 stars, based on 1 article reviews
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    rabbit monoclonal anti gfp - by Bioz Stars, 2024-06
    86/100 stars

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    1) Product Images from "RAPSYN-mediated neddylation of BCR-ABL alternatively determines the fate of Philadelphia chromosome-positive leukemia"

    Article Title: RAPSYN-mediated neddylation of BCR-ABL alternatively determines the fate of Philadelphia chromosome-positive leukemia

    Journal: eLife

    doi: 10.7554/eLife.88375


    Figure Legend Snippet:

    Techniques Used: Transfection, Construct, Plasmid Preparation, Expressing

    chicken anti gfp  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc chicken anti gfp
    Elimination of the X chromosome and creation of DNA double strand break in mouse ES cells by CRISPR/Cas9-mediated gene editing. ( a ) Schematic of X-B, X-C, and X-D target sites and their copies on the mouse X chromosome and representative brightfield microscopy images of mouse ES cells 24 h, 48 h, 72 h, and 144 h after transfection and puromycin selection. Co-transfection with a <t>GFP-expression</t> plasmid was performed to assess transfection efficiency (top). mES cells grow as dome shaped colonies which are apparent at the 144h timepoint. Scale bar = 100 µM. ( b ) Cell survival percentage at 144 h post-transfection of X-shredder gRNAs (n = 3; Bars show mean ± SD, one way ANOVA with Sidak’s multiple comparison test). ( c ) Representative immunofluorescence images of mouse ES cells 24 h after transfection. Scale bar = 10 µM. ( d ) Average numbers <t>of</t> <t>GFP/gamma-H2AX</t> double positive cells (n = 3). Bars show mean ± SD, one way ANOVA with Sidak’s multiple comparison test.
    Chicken Anti Gfp, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/chicken anti gfp/product/Cell Signaling Technology Inc
    Average 86 stars, based on 1 article reviews
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    chicken anti gfp - by Bioz Stars, 2024-06
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    Images

    1) Product Images from "Investigating the potential of X chromosome shredding for mouse genetic biocontrol"

    Article Title: Investigating the potential of X chromosome shredding for mouse genetic biocontrol

    Journal: Scientific Reports

    doi: 10.1038/s41598-024-63706-4

    Elimination of the X chromosome and creation of DNA double strand break in mouse ES cells by CRISPR/Cas9-mediated gene editing. ( a ) Schematic of X-B, X-C, and X-D target sites and their copies on the mouse X chromosome and representative brightfield microscopy images of mouse ES cells 24 h, 48 h, 72 h, and 144 h after transfection and puromycin selection. Co-transfection with a GFP-expression plasmid was performed to assess transfection efficiency (top). mES cells grow as dome shaped colonies which are apparent at the 144h timepoint. Scale bar = 100 µM. ( b ) Cell survival percentage at 144 h post-transfection of X-shredder gRNAs (n = 3; Bars show mean ± SD, one way ANOVA with Sidak’s multiple comparison test). ( c ) Representative immunofluorescence images of mouse ES cells 24 h after transfection. Scale bar = 10 µM. ( d ) Average numbers of GFP/gamma-H2AX double positive cells (n = 3). Bars show mean ± SD, one way ANOVA with Sidak’s multiple comparison test.
    Figure Legend Snippet: Elimination of the X chromosome and creation of DNA double strand break in mouse ES cells by CRISPR/Cas9-mediated gene editing. ( a ) Schematic of X-B, X-C, and X-D target sites and their copies on the mouse X chromosome and representative brightfield microscopy images of mouse ES cells 24 h, 48 h, 72 h, and 144 h after transfection and puromycin selection. Co-transfection with a GFP-expression plasmid was performed to assess transfection efficiency (top). mES cells grow as dome shaped colonies which are apparent at the 144h timepoint. Scale bar = 100 µM. ( b ) Cell survival percentage at 144 h post-transfection of X-shredder gRNAs (n = 3; Bars show mean ± SD, one way ANOVA with Sidak’s multiple comparison test). ( c ) Representative immunofluorescence images of mouse ES cells 24 h after transfection. Scale bar = 10 µM. ( d ) Average numbers of GFP/gamma-H2AX double positive cells (n = 3). Bars show mean ± SD, one way ANOVA with Sidak’s multiple comparison test.

    Techniques Used: CRISPR, Microscopy, Transfection, Selection, Cotransfection, Expressing, Plasmid Preparation, Comparison, Immunofluorescence

    Characterisation of X shredder-mCherry and germline promoter-Cas9 transgenic mice. ( a ) Transgene copy number of hemizygous X-B (n = 2), X-C-1 (n = 3), X-C-2 (n = 3) (two founder lines), and X-D (n = 10) mice. Bars show mean ± SD. ( b ) Fluorescence imaging performed on ear skin punch biopsies for the transgenic mouse lines described in A. showing representative mCherry signal (red) with a WT mCherry negative control. n = > 30 per genotype. Scale bar = 200 µM. ( c ) Expression of Cas9 RNA in testis isolated from Ccna1 -, Prm1 -, and Stra8 -Cas9 transgenic mouse lines. Expression is normalised to eEF2 and WT testis indicates background Cas9 detection in this assay. n = 1 per genotype. ( d ) Representative IF of Cas9-GFP expression (green) in the testis of Ccna1 -Cas9-GFP transgenic mice. GFP signal was amplified by staining with an anti-GFP antibody while DAPI nuclear staining (blue) shows tubule structures. n = 2 per genotype, mice 8–24 weeks of age. Scale bar = 100 µM.
    Figure Legend Snippet: Characterisation of X shredder-mCherry and germline promoter-Cas9 transgenic mice. ( a ) Transgene copy number of hemizygous X-B (n = 2), X-C-1 (n = 3), X-C-2 (n = 3) (two founder lines), and X-D (n = 10) mice. Bars show mean ± SD. ( b ) Fluorescence imaging performed on ear skin punch biopsies for the transgenic mouse lines described in A. showing representative mCherry signal (red) with a WT mCherry negative control. n = > 30 per genotype. Scale bar = 200 µM. ( c ) Expression of Cas9 RNA in testis isolated from Ccna1 -, Prm1 -, and Stra8 -Cas9 transgenic mouse lines. Expression is normalised to eEF2 and WT testis indicates background Cas9 detection in this assay. n = 1 per genotype. ( d ) Representative IF of Cas9-GFP expression (green) in the testis of Ccna1 -Cas9-GFP transgenic mice. GFP signal was amplified by staining with an anti-GFP antibody while DAPI nuclear staining (blue) shows tubule structures. n = 2 per genotype, mice 8–24 weeks of age. Scale bar = 100 µM.

    Techniques Used: Transgenic Assay, Fluorescence, Imaging, Negative Control, Expressing, Isolation, Amplification, Staining

    chicken anti gfp  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc chicken anti gfp
    Chicken Anti Gfp, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    gfp antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc gfp antibody
    (A) Schematic representation of sample preparation and workflow for SiMPull-POP. 1-Membrane fractions <t>containing</t> <t>EphA2-GFP</t> were solubilized with the amphipathic copolymer DIBMA to generate DIBMALPs. 2-EphA2-GFP DIBMALPs were immobilized on a functionalized microscope slide displaying an EphA2 antibody. 3-DIBMALPs devoid of EphA2-GFP are washed away before imaging. (B) Representative single-molecule TIRF image in the presence (left) and absence (right) of EphA2 antibody. Each blue spot represents a DIBMALP containing EphA2-GFP. (C) Representative GFP photobleaching traces showing a stepwise decrease in GFP intensity over time; arrows represent individual photobleaching events. Photobleaching steps are used to infer EphA2 oligomerization status.
    Gfp Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gfp antibody/product/Cell Signaling Technology Inc
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    1) Product Images from "Cholesterol inhibits assembly and activation of the EphA2 receptor"

    Article Title: Cholesterol inhibits assembly and activation of the EphA2 receptor

    Journal: bioRxiv

    doi: 10.1101/2024.06.10.598255

    (A) Schematic representation of sample preparation and workflow for SiMPull-POP. 1-Membrane fractions containing EphA2-GFP were solubilized with the amphipathic copolymer DIBMA to generate DIBMALPs. 2-EphA2-GFP DIBMALPs were immobilized on a functionalized microscope slide displaying an EphA2 antibody. 3-DIBMALPs devoid of EphA2-GFP are washed away before imaging. (B) Representative single-molecule TIRF image in the presence (left) and absence (right) of EphA2 antibody. Each blue spot represents a DIBMALP containing EphA2-GFP. (C) Representative GFP photobleaching traces showing a stepwise decrease in GFP intensity over time; arrows represent individual photobleaching events. Photobleaching steps are used to infer EphA2 oligomerization status.
    Figure Legend Snippet: (A) Schematic representation of sample preparation and workflow for SiMPull-POP. 1-Membrane fractions containing EphA2-GFP were solubilized with the amphipathic copolymer DIBMA to generate DIBMALPs. 2-EphA2-GFP DIBMALPs were immobilized on a functionalized microscope slide displaying an EphA2 antibody. 3-DIBMALPs devoid of EphA2-GFP are washed away before imaging. (B) Representative single-molecule TIRF image in the presence (left) and absence (right) of EphA2 antibody. Each blue spot represents a DIBMALP containing EphA2-GFP. (C) Representative GFP photobleaching traces showing a stepwise decrease in GFP intensity over time; arrows represent individual photobleaching events. Photobleaching steps are used to infer EphA2 oligomerization status.

    Techniques Used: Sample Prep, Membrane, Microscopy, Imaging

    (A) Schematic of DIBMALP containing an EphA2-GFP monomer. (B) Step distribution of control DIBMALPs (black) or those formed from cells treated with EA1 (pink), MβCD (blue) or both (magenta). (C) Oligomeric distribution calculated from data in panel B. (D) Schematic representing DDM micelles containing EphA2-GFP. (E) Step distribution of DDM-solubilized EphA2-GFP in the same conditions as in DIBMALPs. (F) Oligomeric distribution of DDM-solubilized EphA2-GFP photobleaching data. p -values are from two-way ANOVA followed by Tukey multiple comparison test.*, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001.
    Figure Legend Snippet: (A) Schematic of DIBMALP containing an EphA2-GFP monomer. (B) Step distribution of control DIBMALPs (black) or those formed from cells treated with EA1 (pink), MβCD (blue) or both (magenta). (C) Oligomeric distribution calculated from data in panel B. (D) Schematic representing DDM micelles containing EphA2-GFP. (E) Step distribution of DDM-solubilized EphA2-GFP in the same conditions as in DIBMALPs. (F) Oligomeric distribution of DDM-solubilized EphA2-GFP photobleaching data. p -values are from two-way ANOVA followed by Tukey multiple comparison test.*, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001.

    Techniques Used: Comparison

    gfp  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc gfp
    Gfp, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    anti gfp 9f9  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti gfp 9f9
    Anti Gfp 9f9, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti gfp d5 1 rabbit mab
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    Cell Signaling Technology Inc antibody anti mad1 anti gfp or anti myc
    (A) An alphafold model of <t>Mad1-Mad2</t> hetero-tetramer indicating conserved domains in CnMad1: the Mad2 interaction site; 2 putative Bub1 binding motifs (RLK) and the structurally conserved C-terminal domain (RWD). (B) The mad1 Δ strain is benomyl sensitive and can be complemented with an ectopic <t>GFP-Mad1</t> construct. Strains were grown on YPD plates containing the indicated concentrations of benomyl for 3 days at 30°C. (C) Anti-Mad1 immunoblot confirms the mad1 Δ strain. The three strains indicated were grown to log phase, harvested and whole cell extracts generated. The upper band (~115kD, labelled *) recognised by the anti-Mad1 antibody is a cross-reacting band and is used here as a loading control. (D) Benomyl sensitivity of the mad2 Δ and complementation with ectopic GALp-MAD2 . Strains were grown on plates (with 2% glucose or 2% galactose) containing the indicated concentrations of benomyl for 3 days at 30°C. (E) Plate reader experiments confirm that mad1 , mad2 and mps1 mutants are all temperature sensitive, with populations expanding slower than wild-type. Cells were diluted to OD 600 0.2 and grown with shaking at the indicated temperatures. For viability assay (colony forming units) see . (F) The mad1 , mad2 double mutant does not display a synthetic phenotype. mad1 , mad2 , mad1 , mad2 double and mps1 strains were diluted, plated and grown on YPD plates at the indicated temperatures and drug concentrations for 3 days.
    Antibody Anti Mad1 Anti Gfp Or Anti Myc, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Conserved signalling functions for Mps1, Mad1 and Mad2 in the Cryptococcus neoformans spindle checkpoint"

    Article Title: Conserved signalling functions for Mps1, Mad1 and Mad2 in the Cryptococcus neoformans spindle checkpoint

    Journal: PLOS Genetics

    doi: 10.1371/journal.pgen.1011302

    (A) An alphafold model of Mad1-Mad2 hetero-tetramer indicating conserved domains in CnMad1: the Mad2 interaction site; 2 putative Bub1 binding motifs (RLK) and the structurally conserved C-terminal domain (RWD). (B) The mad1 Δ strain is benomyl sensitive and can be complemented with an ectopic GFP-Mad1 construct. Strains were grown on YPD plates containing the indicated concentrations of benomyl for 3 days at 30°C. (C) Anti-Mad1 immunoblot confirms the mad1 Δ strain. The three strains indicated were grown to log phase, harvested and whole cell extracts generated. The upper band (~115kD, labelled *) recognised by the anti-Mad1 antibody is a cross-reacting band and is used here as a loading control. (D) Benomyl sensitivity of the mad2 Δ and complementation with ectopic GALp-MAD2 . Strains were grown on plates (with 2% glucose or 2% galactose) containing the indicated concentrations of benomyl for 3 days at 30°C. (E) Plate reader experiments confirm that mad1 , mad2 and mps1 mutants are all temperature sensitive, with populations expanding slower than wild-type. Cells were diluted to OD 600 0.2 and grown with shaking at the indicated temperatures. For viability assay (colony forming units) see . (F) The mad1 , mad2 double mutant does not display a synthetic phenotype. mad1 , mad2 , mad1 , mad2 double and mps1 strains were diluted, plated and grown on YPD plates at the indicated temperatures and drug concentrations for 3 days.
    Figure Legend Snippet: (A) An alphafold model of Mad1-Mad2 hetero-tetramer indicating conserved domains in CnMad1: the Mad2 interaction site; 2 putative Bub1 binding motifs (RLK) and the structurally conserved C-terminal domain (RWD). (B) The mad1 Δ strain is benomyl sensitive and can be complemented with an ectopic GFP-Mad1 construct. Strains were grown on YPD plates containing the indicated concentrations of benomyl for 3 days at 30°C. (C) Anti-Mad1 immunoblot confirms the mad1 Δ strain. The three strains indicated were grown to log phase, harvested and whole cell extracts generated. The upper band (~115kD, labelled *) recognised by the anti-Mad1 antibody is a cross-reacting band and is used here as a loading control. (D) Benomyl sensitivity of the mad2 Δ and complementation with ectopic GALp-MAD2 . Strains were grown on plates (with 2% glucose or 2% galactose) containing the indicated concentrations of benomyl for 3 days at 30°C. (E) Plate reader experiments confirm that mad1 , mad2 and mps1 mutants are all temperature sensitive, with populations expanding slower than wild-type. Cells were diluted to OD 600 0.2 and grown with shaking at the indicated temperatures. For viability assay (colony forming units) see . (F) The mad1 , mad2 double mutant does not display a synthetic phenotype. mad1 , mad2 , mad1 , mad2 double and mps1 strains were diluted, plated and grown on YPD plates at the indicated temperatures and drug concentrations for 3 days.

    Techniques Used: Binding Assay, Construct, Western Blot, Generated, Viability Assay, Mutagenesis

    (A) mad1 , mad2 and mps1 strains fail to maintain a large-budded nocodazole arrest. The strains indicated were grown to log phase and then nocodazole was added to a final concentration of 2.5 μg/ml in YPD media for 3 hours. Scale bar is 10μm. (B) Quantitation of the large-budded cells (bud diameter >4μm) observed at the 3 hour time point during this nocodazole challenge. This experiment was repeated 3 times and 300 cells were counted for each strain per experiment. (C) Images taken from a microfluidics analysis of this behaviour in nocodazole (for H99, mad1 , mad2 and mps1 mutants). Cells were pre-grown in synthetic complete media supplemented with 0.2g/ml glucose and 0.05% w/v bovine serum albumin (Sigma) and then injected into the microfluidics device and analysed for 4 hours (+/- 2.5μg/ml nocodazole). Scale bar is 10μm. (D) Quantitation of the microfluidics experiment: movies were analysed manually for re-budding. This experiment was repeated three times and at least 100 cells counted per strain each time.
    Figure Legend Snippet: (A) mad1 , mad2 and mps1 strains fail to maintain a large-budded nocodazole arrest. The strains indicated were grown to log phase and then nocodazole was added to a final concentration of 2.5 μg/ml in YPD media for 3 hours. Scale bar is 10μm. (B) Quantitation of the large-budded cells (bud diameter >4μm) observed at the 3 hour time point during this nocodazole challenge. This experiment was repeated 3 times and 300 cells were counted for each strain per experiment. (C) Images taken from a microfluidics analysis of this behaviour in nocodazole (for H99, mad1 , mad2 and mps1 mutants). Cells were pre-grown in synthetic complete media supplemented with 0.2g/ml glucose and 0.05% w/v bovine serum albumin (Sigma) and then injected into the microfluidics device and analysed for 4 hours (+/- 2.5μg/ml nocodazole). Scale bar is 10μm. (D) Quantitation of the microfluidics experiment: movies were analysed manually for re-budding. This experiment was repeated three times and at least 100 cells counted per strain each time.

    Techniques Used: Concentration Assay, Quantitation Assay, Injection

    (A) Strains expressing GFP-Mad1 were stained with Hoechst to label their DNA in cultures grown with and without the addition of 2.5μg/ml nocodazole. Scale bar is 10μm. (B) Five representative stages of mitosis are shown here, from live imaging of a strain expressing both GFP-Mad1 and mCherry-Tub4 (the gamma tubulin construct labels the spindle poles in mitosis). Scale bar is 10μm. (C) In nocodazole arrested cells, GFP-Mad1 is close to but does not co-localise with spindle poles (mCherry-Tub4). Scale bar is 10μm. (D) In nocodazole arrested cells, GFP-Mad1 does co-localise with the centromere marker Cse4-mCherry. Scale bar is 10μm. (E) In interphase, cycling cells, GFP-Mad1 rarely co-localises with Cse4-mCherry. Scale is 10μm.
    Figure Legend Snippet: (A) Strains expressing GFP-Mad1 were stained with Hoechst to label their DNA in cultures grown with and without the addition of 2.5μg/ml nocodazole. Scale bar is 10μm. (B) Five representative stages of mitosis are shown here, from live imaging of a strain expressing both GFP-Mad1 and mCherry-Tub4 (the gamma tubulin construct labels the spindle poles in mitosis). Scale bar is 10μm. (C) In nocodazole arrested cells, GFP-Mad1 is close to but does not co-localise with spindle poles (mCherry-Tub4). Scale bar is 10μm. (D) In nocodazole arrested cells, GFP-Mad1 does co-localise with the centromere marker Cse4-mCherry. Scale bar is 10μm. (E) In interphase, cycling cells, GFP-Mad1 rarely co-localises with Cse4-mCherry. Scale is 10μm.

    Techniques Used: Expressing, Staining, Imaging, Construct, Marker

    GFP-Mad1 was immunoprecipitated from extracts using GFP-TRAP and immunoprecipitates were run into an SDS-PAGE gel, cut out and digested into peptides with trypsin before analysis on a Orbitrap Fusion Lumos Tribrid mass spectrometer. (A) Analysis of GFP-Mad1 interactors: untagged, nocodazole-arrested cells v. nocodazole-arrested GFP-Mad1. This volcano plot shows both the difference (mean, label free quantitation LFQ difference, on the x-axis) and their statistical confidence (-log 10 P-value of Perseus statistical test, on the y-axis) between polypeptides identified in the GFP-Mad1 and untagged control pull-downs (n = 3 for each purification). The -log 10 P-value of 1.3 used here corresponds to a p-value cutoff of 0.05. (B) Analysis of mitotic GFP-Mad1 interactors: cycling GFP-Mad1 cells v. nocodazole-arrested GFP-Mad1. (C) Alignment of the Mad-RLK region in C . neoformans , Homo sapiens , Schizosaccharomyces pombe and Saccharomyces cerevisiae . Only the second RLK (residues 567–9) is within a conserved region. (D) GFP-Mad1 and mCherry-Bub1 co-localise in mitotic cells. Scale bar is 10μm. (E) Co-immunoprecipitation of Mad1 and Bub1 is Mad1-RLK-dependent. mCherry-Bub1 was immunoprecipitated from the five strains indicated. Bub1 immunoprecipitates were washed, split into two sets, separated by SDS-PAGE, then immunoblotted with anti-GFP and anti-Mad1 antibodies.
    Figure Legend Snippet: GFP-Mad1 was immunoprecipitated from extracts using GFP-TRAP and immunoprecipitates were run into an SDS-PAGE gel, cut out and digested into peptides with trypsin before analysis on a Orbitrap Fusion Lumos Tribrid mass spectrometer. (A) Analysis of GFP-Mad1 interactors: untagged, nocodazole-arrested cells v. nocodazole-arrested GFP-Mad1. This volcano plot shows both the difference (mean, label free quantitation LFQ difference, on the x-axis) and their statistical confidence (-log 10 P-value of Perseus statistical test, on the y-axis) between polypeptides identified in the GFP-Mad1 and untagged control pull-downs (n = 3 for each purification). The -log 10 P-value of 1.3 used here corresponds to a p-value cutoff of 0.05. (B) Analysis of mitotic GFP-Mad1 interactors: cycling GFP-Mad1 cells v. nocodazole-arrested GFP-Mad1. (C) Alignment of the Mad-RLK region in C . neoformans , Homo sapiens , Schizosaccharomyces pombe and Saccharomyces cerevisiae . Only the second RLK (residues 567–9) is within a conserved region. (D) GFP-Mad1 and mCherry-Bub1 co-localise in mitotic cells. Scale bar is 10μm. (E) Co-immunoprecipitation of Mad1 and Bub1 is Mad1-RLK-dependent. mCherry-Bub1 was immunoprecipitated from the five strains indicated. Bub1 immunoprecipitates were washed, split into two sets, separated by SDS-PAGE, then immunoblotted with anti-GFP and anti-Mad1 antibodies.

    Techniques Used: Immunoprecipitation, SDS Page, Mass Spectrometry, Quantitation Assay, Purification

    (A) P GAL7 -MPS1 was induced for 5 hours with 2% Galactose, or 2% glucose as the un-induced control. Scale bar is 10μm. (B) Quantitation of this mitotic arrest. Short spindles were scored every hour in at least 100 cells per condition at each time point. This experiment was repeated 3 times and displayed here as the mean +/- confidence levels. (C) Immunoblot of Mps1 levels at each hourly time point after Gal induction. Whole cell extracts were prepared by bead-beating, separated by SDS-PAGE and immunoblotted with the 9E10 anti-myc monoclonal antibody. * indicates a cross-reacting band, used here as a loading control. (D) P GAL7 -MPS1 metaphase arrest is Mad1- and Mad2-dependent. Quantitation of the % large-budded cells with a single, DAPI-stained nucleus in the bud at the 3 hour time point (experiment repeated 3 times and >100 cells counted per sample). (E) Immunoblot of Myc-Mps1 levels in wild-type and mad mutants. Note that the Mps1 protein has reduced gel-mobility in the wild-type extract as these cells are arrested in mitosis where Mps1p is heavily phosphorylated. The myc tag was detected here using the 9B11 monoclonal. * indicates a non-specific band used as a loading control.
    Figure Legend Snippet: (A) P GAL7 -MPS1 was induced for 5 hours with 2% Galactose, or 2% glucose as the un-induced control. Scale bar is 10μm. (B) Quantitation of this mitotic arrest. Short spindles were scored every hour in at least 100 cells per condition at each time point. This experiment was repeated 3 times and displayed here as the mean +/- confidence levels. (C) Immunoblot of Mps1 levels at each hourly time point after Gal induction. Whole cell extracts were prepared by bead-beating, separated by SDS-PAGE and immunoblotted with the 9E10 anti-myc monoclonal antibody. * indicates a cross-reacting band, used here as a loading control. (D) P GAL7 -MPS1 metaphase arrest is Mad1- and Mad2-dependent. Quantitation of the % large-budded cells with a single, DAPI-stained nucleus in the bud at the 3 hour time point (experiment repeated 3 times and >100 cells counted per sample). (E) Immunoblot of Myc-Mps1 levels in wild-type and mad mutants. Note that the Mps1 protein has reduced gel-mobility in the wild-type extract as these cells are arrested in mitosis where Mps1p is heavily phosphorylated. The myc tag was detected here using the 9B11 monoclonal. * indicates a non-specific band used as a loading control.

    Techniques Used: Quantitation Assay, Western Blot, SDS Page, Staining

    (A) Mps1 in vitro kinase assays phosphorylate the Mad1 C-terminus. His-MBP is used here as a negative (specificity) control. Mps1 autophosphorylates and phosphorylates the C-terminus of Mad1. See for phosphopeptides identified in CMad1. (B) Quantitation of the phosphorylation of the Mad1 C-terminus. The radioactive Mad1 signal here was normalised to the Mad1 Coomassie band. (C) Alphafold model of the CnMad1-CTD compared to the crystal structure of hsMad1-CTD. Relevant threonine residues are highlighted on the model. (D) The mad1-T667A mutant, and the double (667,668) phospho-mutant, is benomyl sensitive. The strains indicated were grown at 30°C for 3 days. (E) Anti-GFP immunoblot of whole cell extracts. The GFP- mad1 phospho-mutant proteins are stable. See for a related immunoblot using the anti-Mad1 antibody. (F) The mad1-T667A mutant, and the double phospho-mutant, fails to nocodazole arrest. Cultures were grown to log phase and then challenged with nocodazole for 3 hours. This experiment was repeated 3 times. (G) The mad1-T667A mutant, and the double phospho-mutant, fails to arrest when Mps1 kinase is over-expressed. Log phase cultures were induced with 2% galactose for 5 hours. This experiment was repeated 3 times.
    Figure Legend Snippet: (A) Mps1 in vitro kinase assays phosphorylate the Mad1 C-terminus. His-MBP is used here as a negative (specificity) control. Mps1 autophosphorylates and phosphorylates the C-terminus of Mad1. See for phosphopeptides identified in CMad1. (B) Quantitation of the phosphorylation of the Mad1 C-terminus. The radioactive Mad1 signal here was normalised to the Mad1 Coomassie band. (C) Alphafold model of the CnMad1-CTD compared to the crystal structure of hsMad1-CTD. Relevant threonine residues are highlighted on the model. (D) The mad1-T667A mutant, and the double (667,668) phospho-mutant, is benomyl sensitive. The strains indicated were grown at 30°C for 3 days. (E) Anti-GFP immunoblot of whole cell extracts. The GFP- mad1 phospho-mutant proteins are stable. See for a related immunoblot using the anti-Mad1 antibody. (F) The mad1-T667A mutant, and the double phospho-mutant, fails to nocodazole arrest. Cultures were grown to log phase and then challenged with nocodazole for 3 hours. This experiment was repeated 3 times. (G) The mad1-T667A mutant, and the double phospho-mutant, fails to arrest when Mps1 kinase is over-expressed. Log phase cultures were induced with 2% galactose for 5 hours. This experiment was repeated 3 times.

    Techniques Used: In Vitro, Quantitation Assay, Mutagenesis, Western Blot

    For the full list of strains generated in this study.
    Figure Legend Snippet: For the full list of strains generated in this study.

    Techniques Used: Generated, Selection

    Primers used in this study.
    Figure Legend Snippet: Primers used in this study.

    Techniques Used: Sequencing, Clone Assay, Mutagenesis, Expressing

    Plasmids generated in this study.
    Figure Legend Snippet: Plasmids generated in this study.

    Techniques Used: Generated

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    Cell Signaling Technology Inc rabbit anti gfp
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    (A) An alphafold model of <t>Mad1-Mad2</t> hetero-tetramer indicating conserved domains in CnMad1: the Mad2 interaction site; 2 putative Bub1 binding motifs (RLK) and the structurally conserved C-terminal domain (RWD). (B) The mad1 Δ strain is benomyl sensitive and can be complemented with an ectopic <t>GFP-Mad1</t> construct. Strains were grown on YPD plates containing the indicated concentrations of benomyl for 3 days at 30°C. (C) Anti-Mad1 immunoblot confirms the mad1 Δ strain. The three strains indicated were grown to log phase, harvested and whole cell extracts generated. The upper band (~115kD, labelled *) recognised by the anti-Mad1 antibody is a cross-reacting band and is used here as a loading control. (D) Benomyl sensitivity of the mad2 Δ and complementation with ectopic GALp-MAD2 . Strains were grown on plates (with 2% glucose or 2% galactose) containing the indicated concentrations of benomyl for 3 days at 30°C. (E) Plate reader experiments confirm that mad1 , mad2 and mps1 mutants are all temperature sensitive, with populations expanding slower than wild-type. Cells were diluted to OD 600 0.2 and grown with shaking at the indicated temperatures. For viability assay (colony forming units) see . (F) The mad1 , mad2 double mutant does not display a synthetic phenotype. mad1 , mad2 , mad1 , mad2 double and mps1 strains were diluted, plated and grown on YPD plates at the indicated temperatures and drug concentrations for 3 days.
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    (A) Ccdc66 localizes to the axoneme of the primary cilium. Endogenous staining of Ccdc66 in ciliated IMCD3 cells fixed with 4% PFA was performed using homemade antibody raised against the mouse Ccdc66 protein. Cells were co-stained with anti-acetylated-tubulin, anti-Arl13b and DAPI. The top panel shows images obtained using structured illumination microscopy (SIM). The bottom panel shows confocal microscopy images of samples processed by ultra-structure expansion microscopy (U-ExM). Scale bars SIM: 1 µm, Scale bars U-ExM: 222 nm (B-D) Effects of CCDC66 depletion on cilia formation and length. IMCD3 cells transduced and stably expressing either control or shRNA targeting C-terminus of Ccdc66 (shCcdc66) were grown on glass coverslips, fixed with 4% PFA after 48 h of serum starvation and imaged with confocal microscopy. Scale bar: 5 µm. Insets show 3× magnifications of the cilia, Scale bar: 2 µm (C) Quantification of the percentage of cells with cilia (B). Data represents the mean ± SD of 2 independent experiments. n > 600 cells for control and >400 cells for Ccdc66 depletion. The mean cilia percentage is 84.67% for shControl and 78.72% for shCcdc66. (Welch’s t test, ns: not significant) (D) Quantification of cilia length in 3D, according to acetylated-tubulin signal (co-localizing with Arl13b) (B). The super plot represents the mean ± SEM of 2 independent experiments superimposed onto a scatter plot of normalized individual experimental values to control. The first experimental replicate is shown as a darker circle, and the second as a lighter-colored square. Individual values are represented in lighter shades than the corresponding averages. n > 200 cells for each condition. The mean cilia length in Ccdc66-depleted cells decreased to 0.55-fold compared to the mean control length. (Unpaired t test, *p=0.0114) (E-G) Ccdc66 depletion leads to frequent length fluctuations and enhanced ectocytosis in steady-state cilia. (E) The IMCD3::SSTR3-GFP cells transduced with either control or Ccdc66 shRNA virus were grown in FluoroDish and serum starved for 48 h, then imaged with confocal microscopy with 63× objective. Images were acquired every 4 min for 6 h. Still images are inverted to emphasize cilia better. Scale bar: 3 μm. (F) Ciliary kinetics are presented as normalized length curves, measured from SSTR3-GFP fluorescence in two independent experiments, represented as the mean ± SD. n=20 cilia for both shControl and shCcdc66 conditions. (G) Quantification of ciliary ectocytosis events in (E). Ciliary vesicle or fragment ectocytosis were categorized based on the size of the released EVs, vesicle <500 nm and fragment >500 nm, and plotted as a bar plot. (p values of Welch’s t test, *p=0.0497, **p=0.0095, ns=not significant) Ccdc66, coiled-coil domain-containing protein 66; IMCD3, inner medullary collecting duct cell line; PFA, paraformaldehyde; Arl13b, ADP-ribosylation factor-like protein 13B; DAPI, 4′,6-diamidino-2-phenylindole; shRNA, short hairpin RNA; SSTR3, Somatostatin receptor type 3; GFP, green fluorescent protein; EV, extracellular vesicle; MT, microtubules; DMSO, dimethyl sulfoxide; SEM, standard error of mean; SD, standard deviation.

    Journal: bioRxiv

    Article Title: Ccdc66 regulates primary cilium stability, disassembly and signaling important for epithelial organization

    doi: 10.1101/2024.06.16.599243

    Figure Lengend Snippet: (A) Ccdc66 localizes to the axoneme of the primary cilium. Endogenous staining of Ccdc66 in ciliated IMCD3 cells fixed with 4% PFA was performed using homemade antibody raised against the mouse Ccdc66 protein. Cells were co-stained with anti-acetylated-tubulin, anti-Arl13b and DAPI. The top panel shows images obtained using structured illumination microscopy (SIM). The bottom panel shows confocal microscopy images of samples processed by ultra-structure expansion microscopy (U-ExM). Scale bars SIM: 1 µm, Scale bars U-ExM: 222 nm (B-D) Effects of CCDC66 depletion on cilia formation and length. IMCD3 cells transduced and stably expressing either control or shRNA targeting C-terminus of Ccdc66 (shCcdc66) were grown on glass coverslips, fixed with 4% PFA after 48 h of serum starvation and imaged with confocal microscopy. Scale bar: 5 µm. Insets show 3× magnifications of the cilia, Scale bar: 2 µm (C) Quantification of the percentage of cells with cilia (B). Data represents the mean ± SD of 2 independent experiments. n > 600 cells for control and >400 cells for Ccdc66 depletion. The mean cilia percentage is 84.67% for shControl and 78.72% for shCcdc66. (Welch’s t test, ns: not significant) (D) Quantification of cilia length in 3D, according to acetylated-tubulin signal (co-localizing with Arl13b) (B). The super plot represents the mean ± SEM of 2 independent experiments superimposed onto a scatter plot of normalized individual experimental values to control. The first experimental replicate is shown as a darker circle, and the second as a lighter-colored square. Individual values are represented in lighter shades than the corresponding averages. n > 200 cells for each condition. The mean cilia length in Ccdc66-depleted cells decreased to 0.55-fold compared to the mean control length. (Unpaired t test, *p=0.0114) (E-G) Ccdc66 depletion leads to frequent length fluctuations and enhanced ectocytosis in steady-state cilia. (E) The IMCD3::SSTR3-GFP cells transduced with either control or Ccdc66 shRNA virus were grown in FluoroDish and serum starved for 48 h, then imaged with confocal microscopy with 63× objective. Images were acquired every 4 min for 6 h. Still images are inverted to emphasize cilia better. Scale bar: 3 μm. (F) Ciliary kinetics are presented as normalized length curves, measured from SSTR3-GFP fluorescence in two independent experiments, represented as the mean ± SD. n=20 cilia for both shControl and shCcdc66 conditions. (G) Quantification of ciliary ectocytosis events in (E). Ciliary vesicle or fragment ectocytosis were categorized based on the size of the released EVs, vesicle <500 nm and fragment >500 nm, and plotted as a bar plot. (p values of Welch’s t test, *p=0.0497, **p=0.0095, ns=not significant) Ccdc66, coiled-coil domain-containing protein 66; IMCD3, inner medullary collecting duct cell line; PFA, paraformaldehyde; Arl13b, ADP-ribosylation factor-like protein 13B; DAPI, 4′,6-diamidino-2-phenylindole; shRNA, short hairpin RNA; SSTR3, Somatostatin receptor type 3; GFP, green fluorescent protein; EV, extracellular vesicle; MT, microtubules; DMSO, dimethyl sulfoxide; SEM, standard error of mean; SD, standard deviation.

    Article Snippet: Primary antibodies used for immunoblotting were rat anti-Ccdc66(homemade) at 1:50, mouse anti-Gapdh (CST #97166) at 1:1000, mouse anti-acetylated tubulin (clone 6-11B, 32270, Thermo Fisher) at 1:10000, rabbit anti-GFP at 1:2000 (homemade, previously described in [ ]), anti-Flag (CST #2368) at 1:1000, Streptavidin-IRDye800 coupled at 1:10000.

    Techniques: Staining, Microscopy, Confocal Microscopy, Stable Transfection, Expressing, Control, shRNA, Transduction, Virus, Fluorescence, Standard Deviation

    Live imaging of Ccdc66-depleted cells reveals an increase in ectocytosis and frequent fluctuations in cilia length. (A-C) Cilium disassembly kinetics upon serum stimulation. The IMCD3::SSTR3-GFP cells transduced with either control or Ccdc66 shRNA were grown and serum starved in FluoroDish for 48 h, then imaged with confocal microscopy for 6 h immediately upon serum addition. With images acquired every 4 min. Representative images show one control cilium that gradually resorbs, and three Ccdc66-depleted cilia that undergo disassembly through gradual resorption, instant loss (whole cilium shedding), or a combination of both. Still images are inverted to emphasize cilia better. Scale bar 3 μm (B) Normalized cilia length curves are measured from fluorescence of two independent experiments and represented as the mean ± SD. n=20 cilia for both shControl and shCcdc66 conditions. (C) Quantification of ciliary ectocytosis events in (A). Ciliary events, including vesicle releases (<500 nm) and cilium fragment shedding (>500 nm), were categorized based on the size of the released extracellular vesicles (EVs) and plotted as a bar plot (p values of Welch’s t test *p=0.0272, *p=0.0240, *p=0.0186). (D) Media from IMCD3::SSTR3-GFP was collected 6 h post serum stimulation and separated by high speed centrifugation for subsequent western blot. Pelleted material shows the presence of ciliary markers at expected molecular weights suggesting pelleting of ciliary EVs and fragments. Membranes are blotted with anti-GFP and anti-acetylated tubulin. Shown in the graph is the fold change in the intensity of the acetylated tubulin bands, normalized to shControl of the displayed experimental replicate. The sample was normalized to total cell protein abundance before loading to account for differences in cell numbers between control and Ccdc66-depleted cells. n=2. The bar plot represents mean ± SD of 2 independent experiments. (One sample t test p value **p=0.0087) Ccdc66, coiled-coil domain-containing protein 66; IMCD3, inner medullary collecting duct cell line; SSTR3, Somatostatin receptor type 3; GFP, green fluorescent protein; shRNA, short hairpin RNA; SD, standard deviation.

    Journal: bioRxiv

    Article Title: Ccdc66 regulates primary cilium stability, disassembly and signaling important for epithelial organization

    doi: 10.1101/2024.06.16.599243

    Figure Lengend Snippet: Live imaging of Ccdc66-depleted cells reveals an increase in ectocytosis and frequent fluctuations in cilia length. (A-C) Cilium disassembly kinetics upon serum stimulation. The IMCD3::SSTR3-GFP cells transduced with either control or Ccdc66 shRNA were grown and serum starved in FluoroDish for 48 h, then imaged with confocal microscopy for 6 h immediately upon serum addition. With images acquired every 4 min. Representative images show one control cilium that gradually resorbs, and three Ccdc66-depleted cilia that undergo disassembly through gradual resorption, instant loss (whole cilium shedding), or a combination of both. Still images are inverted to emphasize cilia better. Scale bar 3 μm (B) Normalized cilia length curves are measured from fluorescence of two independent experiments and represented as the mean ± SD. n=20 cilia for both shControl and shCcdc66 conditions. (C) Quantification of ciliary ectocytosis events in (A). Ciliary events, including vesicle releases (<500 nm) and cilium fragment shedding (>500 nm), were categorized based on the size of the released extracellular vesicles (EVs) and plotted as a bar plot (p values of Welch’s t test *p=0.0272, *p=0.0240, *p=0.0186). (D) Media from IMCD3::SSTR3-GFP was collected 6 h post serum stimulation and separated by high speed centrifugation for subsequent western blot. Pelleted material shows the presence of ciliary markers at expected molecular weights suggesting pelleting of ciliary EVs and fragments. Membranes are blotted with anti-GFP and anti-acetylated tubulin. Shown in the graph is the fold change in the intensity of the acetylated tubulin bands, normalized to shControl of the displayed experimental replicate. The sample was normalized to total cell protein abundance before loading to account for differences in cell numbers between control and Ccdc66-depleted cells. n=2. The bar plot represents mean ± SD of 2 independent experiments. (One sample t test p value **p=0.0087) Ccdc66, coiled-coil domain-containing protein 66; IMCD3, inner medullary collecting duct cell line; SSTR3, Somatostatin receptor type 3; GFP, green fluorescent protein; shRNA, short hairpin RNA; SD, standard deviation.

    Article Snippet: Primary antibodies used for immunoblotting were rat anti-Ccdc66(homemade) at 1:50, mouse anti-Gapdh (CST #97166) at 1:1000, mouse anti-acetylated tubulin (clone 6-11B, 32270, Thermo Fisher) at 1:10000, rabbit anti-GFP at 1:2000 (homemade, previously described in [ ]), anti-Flag (CST #2368) at 1:1000, Streptavidin-IRDye800 coupled at 1:10000.

    Techniques: Imaging, Transduction, Control, shRNA, Confocal Microscopy, Fluorescence, Centrifugation, Western Blot, Standard Deviation

    Journal: Genome Medicine

    Article Title: Mitoferrin2 is a synthetic lethal target for chromosome 8p deleted cancers

    doi: 10.1186/s13073-024-01357-w

    Figure Lengend Snippet:

    Article Snippet: GFP , Rabbit , Cell Signaling Technologies , cs2555 , 1:1000.

    Techniques: Western Blot

    Journal: eLife

    Article Title: RAPSYN-mediated neddylation of BCR-ABL alternatively determines the fate of Philadelphia chromosome-positive leukemia

    doi: 10.7554/eLife.88375

    Figure Lengend Snippet:

    Article Snippet: Antibody , Rabbit monoclonal anti-GFP (clone D5.1) , Cell Signaling Technology , Cat# 2956; RRID: AB_1196615 , 1:1000.

    Techniques: Transfection, Construct, Plasmid Preparation, Expressing

    Elimination of the X chromosome and creation of DNA double strand break in mouse ES cells by CRISPR/Cas9-mediated gene editing. ( a ) Schematic of X-B, X-C, and X-D target sites and their copies on the mouse X chromosome and representative brightfield microscopy images of mouse ES cells 24 h, 48 h, 72 h, and 144 h after transfection and puromycin selection. Co-transfection with a GFP-expression plasmid was performed to assess transfection efficiency (top). mES cells grow as dome shaped colonies which are apparent at the 144h timepoint. Scale bar = 100 µM. ( b ) Cell survival percentage at 144 h post-transfection of X-shredder gRNAs (n = 3; Bars show mean ± SD, one way ANOVA with Sidak’s multiple comparison test). ( c ) Representative immunofluorescence images of mouse ES cells 24 h after transfection. Scale bar = 10 µM. ( d ) Average numbers of GFP/gamma-H2AX double positive cells (n = 3). Bars show mean ± SD, one way ANOVA with Sidak’s multiple comparison test.

    Journal: Scientific Reports

    Article Title: Investigating the potential of X chromosome shredding for mouse genetic biocontrol

    doi: 10.1038/s41598-024-63706-4

    Figure Lengend Snippet: Elimination of the X chromosome and creation of DNA double strand break in mouse ES cells by CRISPR/Cas9-mediated gene editing. ( a ) Schematic of X-B, X-C, and X-D target sites and their copies on the mouse X chromosome and representative brightfield microscopy images of mouse ES cells 24 h, 48 h, 72 h, and 144 h after transfection and puromycin selection. Co-transfection with a GFP-expression plasmid was performed to assess transfection efficiency (top). mES cells grow as dome shaped colonies which are apparent at the 144h timepoint. Scale bar = 100 µM. ( b ) Cell survival percentage at 144 h post-transfection of X-shredder gRNAs (n = 3; Bars show mean ± SD, one way ANOVA with Sidak’s multiple comparison test). ( c ) Representative immunofluorescence images of mouse ES cells 24 h after transfection. Scale bar = 10 µM. ( d ) Average numbers of GFP/gamma-H2AX double positive cells (n = 3). Bars show mean ± SD, one way ANOVA with Sidak’s multiple comparison test.

    Article Snippet: Transfected cells were cultured on the gelatin coated coverslips for 24 h. Cells were fixed in 4% paraformaldehyde for 15 min and stained with anti-γ-H2AX (CST,9718) and chicken anti-GFP (Abcam, #ab13970) primary antibodies in 1:400 and 1:600 dilution, respectively.

    Techniques: CRISPR, Microscopy, Transfection, Selection, Cotransfection, Expressing, Plasmid Preparation, Comparison, Immunofluorescence

    Characterisation of X shredder-mCherry and germline promoter-Cas9 transgenic mice. ( a ) Transgene copy number of hemizygous X-B (n = 2), X-C-1 (n = 3), X-C-2 (n = 3) (two founder lines), and X-D (n = 10) mice. Bars show mean ± SD. ( b ) Fluorescence imaging performed on ear skin punch biopsies for the transgenic mouse lines described in A. showing representative mCherry signal (red) with a WT mCherry negative control. n = > 30 per genotype. Scale bar = 200 µM. ( c ) Expression of Cas9 RNA in testis isolated from Ccna1 -, Prm1 -, and Stra8 -Cas9 transgenic mouse lines. Expression is normalised to eEF2 and WT testis indicates background Cas9 detection in this assay. n = 1 per genotype. ( d ) Representative IF of Cas9-GFP expression (green) in the testis of Ccna1 -Cas9-GFP transgenic mice. GFP signal was amplified by staining with an anti-GFP antibody while DAPI nuclear staining (blue) shows tubule structures. n = 2 per genotype, mice 8–24 weeks of age. Scale bar = 100 µM.

    Journal: Scientific Reports

    Article Title: Investigating the potential of X chromosome shredding for mouse genetic biocontrol

    doi: 10.1038/s41598-024-63706-4

    Figure Lengend Snippet: Characterisation of X shredder-mCherry and germline promoter-Cas9 transgenic mice. ( a ) Transgene copy number of hemizygous X-B (n = 2), X-C-1 (n = 3), X-C-2 (n = 3) (two founder lines), and X-D (n = 10) mice. Bars show mean ± SD. ( b ) Fluorescence imaging performed on ear skin punch biopsies for the transgenic mouse lines described in A. showing representative mCherry signal (red) with a WT mCherry negative control. n = > 30 per genotype. Scale bar = 200 µM. ( c ) Expression of Cas9 RNA in testis isolated from Ccna1 -, Prm1 -, and Stra8 -Cas9 transgenic mouse lines. Expression is normalised to eEF2 and WT testis indicates background Cas9 detection in this assay. n = 1 per genotype. ( d ) Representative IF of Cas9-GFP expression (green) in the testis of Ccna1 -Cas9-GFP transgenic mice. GFP signal was amplified by staining with an anti-GFP antibody while DAPI nuclear staining (blue) shows tubule structures. n = 2 per genotype, mice 8–24 weeks of age. Scale bar = 100 µM.

    Article Snippet: Transfected cells were cultured on the gelatin coated coverslips for 24 h. Cells were fixed in 4% paraformaldehyde for 15 min and stained with anti-γ-H2AX (CST,9718) and chicken anti-GFP (Abcam, #ab13970) primary antibodies in 1:400 and 1:600 dilution, respectively.

    Techniques: Transgenic Assay, Fluorescence, Imaging, Negative Control, Expressing, Isolation, Amplification, Staining

    (A) Schematic representation of sample preparation and workflow for SiMPull-POP. 1-Membrane fractions containing EphA2-GFP were solubilized with the amphipathic copolymer DIBMA to generate DIBMALPs. 2-EphA2-GFP DIBMALPs were immobilized on a functionalized microscope slide displaying an EphA2 antibody. 3-DIBMALPs devoid of EphA2-GFP are washed away before imaging. (B) Representative single-molecule TIRF image in the presence (left) and absence (right) of EphA2 antibody. Each blue spot represents a DIBMALP containing EphA2-GFP. (C) Representative GFP photobleaching traces showing a stepwise decrease in GFP intensity over time; arrows represent individual photobleaching events. Photobleaching steps are used to infer EphA2 oligomerization status.

    Journal: bioRxiv

    Article Title: Cholesterol inhibits assembly and activation of the EphA2 receptor

    doi: 10.1101/2024.06.10.598255

    Figure Lengend Snippet: (A) Schematic representation of sample preparation and workflow for SiMPull-POP. 1-Membrane fractions containing EphA2-GFP were solubilized with the amphipathic copolymer DIBMA to generate DIBMALPs. 2-EphA2-GFP DIBMALPs were immobilized on a functionalized microscope slide displaying an EphA2 antibody. 3-DIBMALPs devoid of EphA2-GFP are washed away before imaging. (B) Representative single-molecule TIRF image in the presence (left) and absence (right) of EphA2 antibody. Each blue spot represents a DIBMALP containing EphA2-GFP. (C) Representative GFP photobleaching traces showing a stepwise decrease in GFP intensity over time; arrows represent individual photobleaching events. Photobleaching steps are used to infer EphA2 oligomerization status.

    Article Snippet: To immobilize protein samples, 0.02 mg/mL NeutrAvidin protein (Thermo Fisher Scientific) was first incubated in the chamber for 10 minutes followed by the addition of a biotinylated EphA2 or GFP antibody for 20 minutes (Cell Signaling and Rockland Immunochemical Inc., respectively).

    Techniques: Sample Prep, Membrane, Microscopy, Imaging

    (A) Schematic of DIBMALP containing an EphA2-GFP monomer. (B) Step distribution of control DIBMALPs (black) or those formed from cells treated with EA1 (pink), MβCD (blue) or both (magenta). (C) Oligomeric distribution calculated from data in panel B. (D) Schematic representing DDM micelles containing EphA2-GFP. (E) Step distribution of DDM-solubilized EphA2-GFP in the same conditions as in DIBMALPs. (F) Oligomeric distribution of DDM-solubilized EphA2-GFP photobleaching data. p -values are from two-way ANOVA followed by Tukey multiple comparison test.*, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001.

    Journal: bioRxiv

    Article Title: Cholesterol inhibits assembly and activation of the EphA2 receptor

    doi: 10.1101/2024.06.10.598255

    Figure Lengend Snippet: (A) Schematic of DIBMALP containing an EphA2-GFP monomer. (B) Step distribution of control DIBMALPs (black) or those formed from cells treated with EA1 (pink), MβCD (blue) or both (magenta). (C) Oligomeric distribution calculated from data in panel B. (D) Schematic representing DDM micelles containing EphA2-GFP. (E) Step distribution of DDM-solubilized EphA2-GFP in the same conditions as in DIBMALPs. (F) Oligomeric distribution of DDM-solubilized EphA2-GFP photobleaching data. p -values are from two-way ANOVA followed by Tukey multiple comparison test.*, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001.

    Article Snippet: To immobilize protein samples, 0.02 mg/mL NeutrAvidin protein (Thermo Fisher Scientific) was first incubated in the chamber for 10 minutes followed by the addition of a biotinylated EphA2 or GFP antibody for 20 minutes (Cell Signaling and Rockland Immunochemical Inc., respectively).

    Techniques: Comparison

    (A) An alphafold model of Mad1-Mad2 hetero-tetramer indicating conserved domains in CnMad1: the Mad2 interaction site; 2 putative Bub1 binding motifs (RLK) and the structurally conserved C-terminal domain (RWD). (B) The mad1 Δ strain is benomyl sensitive and can be complemented with an ectopic GFP-Mad1 construct. Strains were grown on YPD plates containing the indicated concentrations of benomyl for 3 days at 30°C. (C) Anti-Mad1 immunoblot confirms the mad1 Δ strain. The three strains indicated were grown to log phase, harvested and whole cell extracts generated. The upper band (~115kD, labelled *) recognised by the anti-Mad1 antibody is a cross-reacting band and is used here as a loading control. (D) Benomyl sensitivity of the mad2 Δ and complementation with ectopic GALp-MAD2 . Strains were grown on plates (with 2% glucose or 2% galactose) containing the indicated concentrations of benomyl for 3 days at 30°C. (E) Plate reader experiments confirm that mad1 , mad2 and mps1 mutants are all temperature sensitive, with populations expanding slower than wild-type. Cells were diluted to OD 600 0.2 and grown with shaking at the indicated temperatures. For viability assay (colony forming units) see . (F) The mad1 , mad2 double mutant does not display a synthetic phenotype. mad1 , mad2 , mad1 , mad2 double and mps1 strains were diluted, plated and grown on YPD plates at the indicated temperatures and drug concentrations for 3 days.

    Journal: PLOS Genetics

    Article Title: Conserved signalling functions for Mps1, Mad1 and Mad2 in the Cryptococcus neoformans spindle checkpoint

    doi: 10.1371/journal.pgen.1011302

    Figure Lengend Snippet: (A) An alphafold model of Mad1-Mad2 hetero-tetramer indicating conserved domains in CnMad1: the Mad2 interaction site; 2 putative Bub1 binding motifs (RLK) and the structurally conserved C-terminal domain (RWD). (B) The mad1 Δ strain is benomyl sensitive and can be complemented with an ectopic GFP-Mad1 construct. Strains were grown on YPD plates containing the indicated concentrations of benomyl for 3 days at 30°C. (C) Anti-Mad1 immunoblot confirms the mad1 Δ strain. The three strains indicated were grown to log phase, harvested and whole cell extracts generated. The upper band (~115kD, labelled *) recognised by the anti-Mad1 antibody is a cross-reacting band and is used here as a loading control. (D) Benomyl sensitivity of the mad2 Δ and complementation with ectopic GALp-MAD2 . Strains were grown on plates (with 2% glucose or 2% galactose) containing the indicated concentrations of benomyl for 3 days at 30°C. (E) Plate reader experiments confirm that mad1 , mad2 and mps1 mutants are all temperature sensitive, with populations expanding slower than wild-type. Cells were diluted to OD 600 0.2 and grown with shaking at the indicated temperatures. For viability assay (colony forming units) see . (F) The mad1 , mad2 double mutant does not display a synthetic phenotype. mad1 , mad2 , mad1 , mad2 double and mps1 strains were diluted, plated and grown on YPD plates at the indicated temperatures and drug concentrations for 3 days.

    Article Snippet: The primary antibody (anti-Mad1, anti-GFP or anti-Myc [9E10 or 9B11, Cell Signalling]) was incubated in the same blocking buffer (1 in 1000 dilution) overnight at 4°C.

    Techniques: Binding Assay, Construct, Western Blot, Generated, Viability Assay, Mutagenesis

    (A) mad1 , mad2 and mps1 strains fail to maintain a large-budded nocodazole arrest. The strains indicated were grown to log phase and then nocodazole was added to a final concentration of 2.5 μg/ml in YPD media for 3 hours. Scale bar is 10μm. (B) Quantitation of the large-budded cells (bud diameter >4μm) observed at the 3 hour time point during this nocodazole challenge. This experiment was repeated 3 times and 300 cells were counted for each strain per experiment. (C) Images taken from a microfluidics analysis of this behaviour in nocodazole (for H99, mad1 , mad2 and mps1 mutants). Cells were pre-grown in synthetic complete media supplemented with 0.2g/ml glucose and 0.05% w/v bovine serum albumin (Sigma) and then injected into the microfluidics device and analysed for 4 hours (+/- 2.5μg/ml nocodazole). Scale bar is 10μm. (D) Quantitation of the microfluidics experiment: movies were analysed manually for re-budding. This experiment was repeated three times and at least 100 cells counted per strain each time.

    Journal: PLOS Genetics

    Article Title: Conserved signalling functions for Mps1, Mad1 and Mad2 in the Cryptococcus neoformans spindle checkpoint

    doi: 10.1371/journal.pgen.1011302

    Figure Lengend Snippet: (A) mad1 , mad2 and mps1 strains fail to maintain a large-budded nocodazole arrest. The strains indicated were grown to log phase and then nocodazole was added to a final concentration of 2.5 μg/ml in YPD media for 3 hours. Scale bar is 10μm. (B) Quantitation of the large-budded cells (bud diameter >4μm) observed at the 3 hour time point during this nocodazole challenge. This experiment was repeated 3 times and 300 cells were counted for each strain per experiment. (C) Images taken from a microfluidics analysis of this behaviour in nocodazole (for H99, mad1 , mad2 and mps1 mutants). Cells were pre-grown in synthetic complete media supplemented with 0.2g/ml glucose and 0.05% w/v bovine serum albumin (Sigma) and then injected into the microfluidics device and analysed for 4 hours (+/- 2.5μg/ml nocodazole). Scale bar is 10μm. (D) Quantitation of the microfluidics experiment: movies were analysed manually for re-budding. This experiment was repeated three times and at least 100 cells counted per strain each time.

    Article Snippet: The primary antibody (anti-Mad1, anti-GFP or anti-Myc [9E10 or 9B11, Cell Signalling]) was incubated in the same blocking buffer (1 in 1000 dilution) overnight at 4°C.

    Techniques: Concentration Assay, Quantitation Assay, Injection

    (A) Strains expressing GFP-Mad1 were stained with Hoechst to label their DNA in cultures grown with and without the addition of 2.5μg/ml nocodazole. Scale bar is 10μm. (B) Five representative stages of mitosis are shown here, from live imaging of a strain expressing both GFP-Mad1 and mCherry-Tub4 (the gamma tubulin construct labels the spindle poles in mitosis). Scale bar is 10μm. (C) In nocodazole arrested cells, GFP-Mad1 is close to but does not co-localise with spindle poles (mCherry-Tub4). Scale bar is 10μm. (D) In nocodazole arrested cells, GFP-Mad1 does co-localise with the centromere marker Cse4-mCherry. Scale bar is 10μm. (E) In interphase, cycling cells, GFP-Mad1 rarely co-localises with Cse4-mCherry. Scale is 10μm.

    Journal: PLOS Genetics

    Article Title: Conserved signalling functions for Mps1, Mad1 and Mad2 in the Cryptococcus neoformans spindle checkpoint

    doi: 10.1371/journal.pgen.1011302

    Figure Lengend Snippet: (A) Strains expressing GFP-Mad1 were stained with Hoechst to label their DNA in cultures grown with and without the addition of 2.5μg/ml nocodazole. Scale bar is 10μm. (B) Five representative stages of mitosis are shown here, from live imaging of a strain expressing both GFP-Mad1 and mCherry-Tub4 (the gamma tubulin construct labels the spindle poles in mitosis). Scale bar is 10μm. (C) In nocodazole arrested cells, GFP-Mad1 is close to but does not co-localise with spindle poles (mCherry-Tub4). Scale bar is 10μm. (D) In nocodazole arrested cells, GFP-Mad1 does co-localise with the centromere marker Cse4-mCherry. Scale bar is 10μm. (E) In interphase, cycling cells, GFP-Mad1 rarely co-localises with Cse4-mCherry. Scale is 10μm.

    Article Snippet: The primary antibody (anti-Mad1, anti-GFP or anti-Myc [9E10 or 9B11, Cell Signalling]) was incubated in the same blocking buffer (1 in 1000 dilution) overnight at 4°C.

    Techniques: Expressing, Staining, Imaging, Construct, Marker

    GFP-Mad1 was immunoprecipitated from extracts using GFP-TRAP and immunoprecipitates were run into an SDS-PAGE gel, cut out and digested into peptides with trypsin before analysis on a Orbitrap Fusion Lumos Tribrid mass spectrometer. (A) Analysis of GFP-Mad1 interactors: untagged, nocodazole-arrested cells v. nocodazole-arrested GFP-Mad1. This volcano plot shows both the difference (mean, label free quantitation LFQ difference, on the x-axis) and their statistical confidence (-log 10 P-value of Perseus statistical test, on the y-axis) between polypeptides identified in the GFP-Mad1 and untagged control pull-downs (n = 3 for each purification). The -log 10 P-value of 1.3 used here corresponds to a p-value cutoff of 0.05. (B) Analysis of mitotic GFP-Mad1 interactors: cycling GFP-Mad1 cells v. nocodazole-arrested GFP-Mad1. (C) Alignment of the Mad-RLK region in C . neoformans , Homo sapiens , Schizosaccharomyces pombe and Saccharomyces cerevisiae . Only the second RLK (residues 567–9) is within a conserved region. (D) GFP-Mad1 and mCherry-Bub1 co-localise in mitotic cells. Scale bar is 10μm. (E) Co-immunoprecipitation of Mad1 and Bub1 is Mad1-RLK-dependent. mCherry-Bub1 was immunoprecipitated from the five strains indicated. Bub1 immunoprecipitates were washed, split into two sets, separated by SDS-PAGE, then immunoblotted with anti-GFP and anti-Mad1 antibodies.

    Journal: PLOS Genetics

    Article Title: Conserved signalling functions for Mps1, Mad1 and Mad2 in the Cryptococcus neoformans spindle checkpoint

    doi: 10.1371/journal.pgen.1011302

    Figure Lengend Snippet: GFP-Mad1 was immunoprecipitated from extracts using GFP-TRAP and immunoprecipitates were run into an SDS-PAGE gel, cut out and digested into peptides with trypsin before analysis on a Orbitrap Fusion Lumos Tribrid mass spectrometer. (A) Analysis of GFP-Mad1 interactors: untagged, nocodazole-arrested cells v. nocodazole-arrested GFP-Mad1. This volcano plot shows both the difference (mean, label free quantitation LFQ difference, on the x-axis) and their statistical confidence (-log 10 P-value of Perseus statistical test, on the y-axis) between polypeptides identified in the GFP-Mad1 and untagged control pull-downs (n = 3 for each purification). The -log 10 P-value of 1.3 used here corresponds to a p-value cutoff of 0.05. (B) Analysis of mitotic GFP-Mad1 interactors: cycling GFP-Mad1 cells v. nocodazole-arrested GFP-Mad1. (C) Alignment of the Mad-RLK region in C . neoformans , Homo sapiens , Schizosaccharomyces pombe and Saccharomyces cerevisiae . Only the second RLK (residues 567–9) is within a conserved region. (D) GFP-Mad1 and mCherry-Bub1 co-localise in mitotic cells. Scale bar is 10μm. (E) Co-immunoprecipitation of Mad1 and Bub1 is Mad1-RLK-dependent. mCherry-Bub1 was immunoprecipitated from the five strains indicated. Bub1 immunoprecipitates were washed, split into two sets, separated by SDS-PAGE, then immunoblotted with anti-GFP and anti-Mad1 antibodies.

    Article Snippet: The primary antibody (anti-Mad1, anti-GFP or anti-Myc [9E10 or 9B11, Cell Signalling]) was incubated in the same blocking buffer (1 in 1000 dilution) overnight at 4°C.

    Techniques: Immunoprecipitation, SDS Page, Mass Spectrometry, Quantitation Assay, Purification

    (A) P GAL7 -MPS1 was induced for 5 hours with 2% Galactose, or 2% glucose as the un-induced control. Scale bar is 10μm. (B) Quantitation of this mitotic arrest. Short spindles were scored every hour in at least 100 cells per condition at each time point. This experiment was repeated 3 times and displayed here as the mean +/- confidence levels. (C) Immunoblot of Mps1 levels at each hourly time point after Gal induction. Whole cell extracts were prepared by bead-beating, separated by SDS-PAGE and immunoblotted with the 9E10 anti-myc monoclonal antibody. * indicates a cross-reacting band, used here as a loading control. (D) P GAL7 -MPS1 metaphase arrest is Mad1- and Mad2-dependent. Quantitation of the % large-budded cells with a single, DAPI-stained nucleus in the bud at the 3 hour time point (experiment repeated 3 times and >100 cells counted per sample). (E) Immunoblot of Myc-Mps1 levels in wild-type and mad mutants. Note that the Mps1 protein has reduced gel-mobility in the wild-type extract as these cells are arrested in mitosis where Mps1p is heavily phosphorylated. The myc tag was detected here using the 9B11 monoclonal. * indicates a non-specific band used as a loading control.

    Journal: PLOS Genetics

    Article Title: Conserved signalling functions for Mps1, Mad1 and Mad2 in the Cryptococcus neoformans spindle checkpoint

    doi: 10.1371/journal.pgen.1011302

    Figure Lengend Snippet: (A) P GAL7 -MPS1 was induced for 5 hours with 2% Galactose, or 2% glucose as the un-induced control. Scale bar is 10μm. (B) Quantitation of this mitotic arrest. Short spindles were scored every hour in at least 100 cells per condition at each time point. This experiment was repeated 3 times and displayed here as the mean +/- confidence levels. (C) Immunoblot of Mps1 levels at each hourly time point after Gal induction. Whole cell extracts were prepared by bead-beating, separated by SDS-PAGE and immunoblotted with the 9E10 anti-myc monoclonal antibody. * indicates a cross-reacting band, used here as a loading control. (D) P GAL7 -MPS1 metaphase arrest is Mad1- and Mad2-dependent. Quantitation of the % large-budded cells with a single, DAPI-stained nucleus in the bud at the 3 hour time point (experiment repeated 3 times and >100 cells counted per sample). (E) Immunoblot of Myc-Mps1 levels in wild-type and mad mutants. Note that the Mps1 protein has reduced gel-mobility in the wild-type extract as these cells are arrested in mitosis where Mps1p is heavily phosphorylated. The myc tag was detected here using the 9B11 monoclonal. * indicates a non-specific band used as a loading control.

    Article Snippet: The primary antibody (anti-Mad1, anti-GFP or anti-Myc [9E10 or 9B11, Cell Signalling]) was incubated in the same blocking buffer (1 in 1000 dilution) overnight at 4°C.

    Techniques: Quantitation Assay, Western Blot, SDS Page, Staining

    (A) Mps1 in vitro kinase assays phosphorylate the Mad1 C-terminus. His-MBP is used here as a negative (specificity) control. Mps1 autophosphorylates and phosphorylates the C-terminus of Mad1. See for phosphopeptides identified in CMad1. (B) Quantitation of the phosphorylation of the Mad1 C-terminus. The radioactive Mad1 signal here was normalised to the Mad1 Coomassie band. (C) Alphafold model of the CnMad1-CTD compared to the crystal structure of hsMad1-CTD. Relevant threonine residues are highlighted on the model. (D) The mad1-T667A mutant, and the double (667,668) phospho-mutant, is benomyl sensitive. The strains indicated were grown at 30°C for 3 days. (E) Anti-GFP immunoblot of whole cell extracts. The GFP- mad1 phospho-mutant proteins are stable. See for a related immunoblot using the anti-Mad1 antibody. (F) The mad1-T667A mutant, and the double phospho-mutant, fails to nocodazole arrest. Cultures were grown to log phase and then challenged with nocodazole for 3 hours. This experiment was repeated 3 times. (G) The mad1-T667A mutant, and the double phospho-mutant, fails to arrest when Mps1 kinase is over-expressed. Log phase cultures were induced with 2% galactose for 5 hours. This experiment was repeated 3 times.

    Journal: PLOS Genetics

    Article Title: Conserved signalling functions for Mps1, Mad1 and Mad2 in the Cryptococcus neoformans spindle checkpoint

    doi: 10.1371/journal.pgen.1011302

    Figure Lengend Snippet: (A) Mps1 in vitro kinase assays phosphorylate the Mad1 C-terminus. His-MBP is used here as a negative (specificity) control. Mps1 autophosphorylates and phosphorylates the C-terminus of Mad1. See for phosphopeptides identified in CMad1. (B) Quantitation of the phosphorylation of the Mad1 C-terminus. The radioactive Mad1 signal here was normalised to the Mad1 Coomassie band. (C) Alphafold model of the CnMad1-CTD compared to the crystal structure of hsMad1-CTD. Relevant threonine residues are highlighted on the model. (D) The mad1-T667A mutant, and the double (667,668) phospho-mutant, is benomyl sensitive. The strains indicated were grown at 30°C for 3 days. (E) Anti-GFP immunoblot of whole cell extracts. The GFP- mad1 phospho-mutant proteins are stable. See for a related immunoblot using the anti-Mad1 antibody. (F) The mad1-T667A mutant, and the double phospho-mutant, fails to nocodazole arrest. Cultures were grown to log phase and then challenged with nocodazole for 3 hours. This experiment was repeated 3 times. (G) The mad1-T667A mutant, and the double phospho-mutant, fails to arrest when Mps1 kinase is over-expressed. Log phase cultures were induced with 2% galactose for 5 hours. This experiment was repeated 3 times.

    Article Snippet: The primary antibody (anti-Mad1, anti-GFP or anti-Myc [9E10 or 9B11, Cell Signalling]) was incubated in the same blocking buffer (1 in 1000 dilution) overnight at 4°C.

    Techniques: In Vitro, Quantitation Assay, Mutagenesis, Western Blot

    For the full list of strains generated in this study.

    Journal: PLOS Genetics

    Article Title: Conserved signalling functions for Mps1, Mad1 and Mad2 in the Cryptococcus neoformans spindle checkpoint

    doi: 10.1371/journal.pgen.1011302

    Figure Lengend Snippet: For the full list of strains generated in this study.

    Article Snippet: The primary antibody (anti-Mad1, anti-GFP or anti-Myc [9E10 or 9B11, Cell Signalling]) was incubated in the same blocking buffer (1 in 1000 dilution) overnight at 4°C.

    Techniques: Generated, Selection

    Primers used in this study.

    Journal: PLOS Genetics

    Article Title: Conserved signalling functions for Mps1, Mad1 and Mad2 in the Cryptococcus neoformans spindle checkpoint

    doi: 10.1371/journal.pgen.1011302

    Figure Lengend Snippet: Primers used in this study.

    Article Snippet: The primary antibody (anti-Mad1, anti-GFP or anti-Myc [9E10 or 9B11, Cell Signalling]) was incubated in the same blocking buffer (1 in 1000 dilution) overnight at 4°C.

    Techniques: Sequencing, Clone Assay, Mutagenesis, Expressing

    Plasmids generated in this study.

    Journal: PLOS Genetics

    Article Title: Conserved signalling functions for Mps1, Mad1 and Mad2 in the Cryptococcus neoformans spindle checkpoint

    doi: 10.1371/journal.pgen.1011302

    Figure Lengend Snippet: Plasmids generated in this study.

    Article Snippet: The primary antibody (anti-Mad1, anti-GFP or anti-Myc [9E10 or 9B11, Cell Signalling]) was incubated in the same blocking buffer (1 in 1000 dilution) overnight at 4°C.

    Techniques: Generated