mouse anti β actin polyclonal antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc mouse anti β actin polyclonal antibody
    Mouse Anti β Actin Polyclonal Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse anti β actin polyclonal antibody/product/Cell Signaling Technology Inc
    Average 96 stars, based on 1 article reviews
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    mouse anti β actin polyclonal antibody - by Bioz Stars, 2023-03
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    primary polyclonal antibodies targeting anti yap1  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc primary polyclonal antibodies targeting anti yap1
    (A and B) Representative images and analysis of immunohistochemistry staining for <t>YAP1</t> (×100) and YAP1(s127) (×200) in human HCC subgrouped as shown in the figure. ImageJ was used to analyze the images. (C–E) Linear regression analysis of the expression of RP11-40C6.2 and YAP1 (IOD value) or percentage of YAP1 nuclear translocation and YAP1 (s127) in human HCC. (F) Diagram of potential TEADs binding sites and mutation design within the RP11-40C6.2 promoter region. (G) Luciferase reporter assay of HBx, HBc, and LPA treatment on the activity of wild-type (WT) and TEADs binding site mutation (Mut) RP11-40C6.2 promoter. (H) Antibody to TEAD 1-4 was used in chromatin immunoprecipitation (ChIP) assays; p65 and IgG were negative controls. (I) Co-IP assays were performed to study the binding between TAZ and HBx or HBc; the protein complex was immuno-precipitated with TAZ antibody and further examined by HBx or HBc antibody. Mean±SEM values from at least three independent experiments are presented. * p <0.05, ** p <0.01.
    Primary Polyclonal Antibodies Targeting Anti Yap1, 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/primary polyclonal antibodies targeting anti yap1/product/Cell Signaling Technology Inc
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    Images

    1) Product Images from "RP11-40C6.2 Inactivates Hippo Signaling by Attenuating YAP1 Ubiquitylation in Hepatitis B Virus-associated Hepatocellular Carcinoma"

    Article Title: RP11-40C6.2 Inactivates Hippo Signaling by Attenuating YAP1 Ubiquitylation in Hepatitis B Virus-associated Hepatocellular Carcinoma

    Journal: Journal of Clinical and Translational Hepatology

    doi: 10.14218/JCTH.2021.00584

    (A and B) Representative images and analysis of immunohistochemistry staining for YAP1 (×100) and YAP1(s127) (×200) in human HCC subgrouped as shown in the figure. ImageJ was used to analyze the images. (C–E) Linear regression analysis of the expression of RP11-40C6.2 and YAP1 (IOD value) or percentage of YAP1 nuclear translocation and YAP1 (s127) in human HCC. (F) Diagram of potential TEADs binding sites and mutation design within the RP11-40C6.2 promoter region. (G) Luciferase reporter assay of HBx, HBc, and LPA treatment on the activity of wild-type (WT) and TEADs binding site mutation (Mut) RP11-40C6.2 promoter. (H) Antibody to TEAD 1-4 was used in chromatin immunoprecipitation (ChIP) assays; p65 and IgG were negative controls. (I) Co-IP assays were performed to study the binding between TAZ and HBx or HBc; the protein complex was immuno-precipitated with TAZ antibody and further examined by HBx or HBc antibody. Mean±SEM values from at least three independent experiments are presented. * p <0.05, ** p <0.01.
    Figure Legend Snippet: (A and B) Representative images and analysis of immunohistochemistry staining for YAP1 (×100) and YAP1(s127) (×200) in human HCC subgrouped as shown in the figure. ImageJ was used to analyze the images. (C–E) Linear regression analysis of the expression of RP11-40C6.2 and YAP1 (IOD value) or percentage of YAP1 nuclear translocation and YAP1 (s127) in human HCC. (F) Diagram of potential TEADs binding sites and mutation design within the RP11-40C6.2 promoter region. (G) Luciferase reporter assay of HBx, HBc, and LPA treatment on the activity of wild-type (WT) and TEADs binding site mutation (Mut) RP11-40C6.2 promoter. (H) Antibody to TEAD 1-4 was used in chromatin immunoprecipitation (ChIP) assays; p65 and IgG were negative controls. (I) Co-IP assays were performed to study the binding between TAZ and HBx or HBc; the protein complex was immuno-precipitated with TAZ antibody and further examined by HBx or HBc antibody. Mean±SEM values from at least three independent experiments are presented. * p <0.05, ** p <0.01.

    Techniques Used: Immunohistochemistry, Staining, Expressing, Translocation Assay, Binding Assay, Mutagenesis, Luciferase, Reporter Assay, Activity Assay, Chromatin Immunoprecipitation, Co-Immunoprecipitation Assay

    (A) Real-time PCR in SMMC-7721, MHCC-97H, Huh7, and Hep3B cells shows the expression of RP11-40C6.2. (B) CCK8 assays show the proliferation of SMMC-7721, MHCC-97H, Huh7, and Hep3B cells. (C) Transwell assays show the invasiveness of SMMC-7721, MHCC-97H, Huh7, and Hep3B cells. (D–F) The expression of RP11-40C6.2 and proliferation and invasiveness of Hep3B, SMMC-7721-RP11-40C6.2, Hep3B, and Hep3B-RP11-40C6.2 shRNA. (G, H) Results of CCK8 and Transwell assays of proliferation and invasiveness of SMMC-7721-RP11-40C6.2 and Hep3B cells treated with or without CA3. (I–J) HCC cell line was treated with or without LPA (0.1 µM) and protein expression of YAP1(s127) was assayed by western blotting with beta actin as an internal control. Mean±SEM values from at least three independent experiments are shown. * p <0.05, ** p <0.01.
    Figure Legend Snippet: (A) Real-time PCR in SMMC-7721, MHCC-97H, Huh7, and Hep3B cells shows the expression of RP11-40C6.2. (B) CCK8 assays show the proliferation of SMMC-7721, MHCC-97H, Huh7, and Hep3B cells. (C) Transwell assays show the invasiveness of SMMC-7721, MHCC-97H, Huh7, and Hep3B cells. (D–F) The expression of RP11-40C6.2 and proliferation and invasiveness of Hep3B, SMMC-7721-RP11-40C6.2, Hep3B, and Hep3B-RP11-40C6.2 shRNA. (G, H) Results of CCK8 and Transwell assays of proliferation and invasiveness of SMMC-7721-RP11-40C6.2 and Hep3B cells treated with or without CA3. (I–J) HCC cell line was treated with or without LPA (0.1 µM) and protein expression of YAP1(s127) was assayed by western blotting with beta actin as an internal control. Mean±SEM values from at least three independent experiments are shown. * p <0.05, ** p <0.01.

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing, shRNA, Western Blot

    (A, B) catRAPID was used to predict hidden binding sites of RP11-40C6.2 and YAP1. The potential binding regions and interaction marks are shown. (C) RNA pull-down performed with sense and antisense probes specific for RP11-40C6.2 and controlled by beads. Western blotting detected precipitated and input total proteins with YAP1 antibody. (D) Ubiquitination detection of YAP1 in SMMC-7721 and Hep3B cells transfected with WT and MU RP11-40C6.2. (E) Proliferation of SMMC-7721 and Hep3B cells transfected with WT and MU RP11-40C6.2. (F) IF staining assay was performed to detect YAP1 nuclear translocation in SMMC-7721 and Hep3B cells transfected with WT and MU RP11-40C6.2. (G) Transwell cell migration assay was used to assess invasion of SMMC-7721 and Hep3B cells transfected with WT and MU RP11-40C6.2. Mean±SEM values from at least three independent experiments are presented. * p <0.05, ** p <0.01.
    Figure Legend Snippet: (A, B) catRAPID was used to predict hidden binding sites of RP11-40C6.2 and YAP1. The potential binding regions and interaction marks are shown. (C) RNA pull-down performed with sense and antisense probes specific for RP11-40C6.2 and controlled by beads. Western blotting detected precipitated and input total proteins with YAP1 antibody. (D) Ubiquitination detection of YAP1 in SMMC-7721 and Hep3B cells transfected with WT and MU RP11-40C6.2. (E) Proliferation of SMMC-7721 and Hep3B cells transfected with WT and MU RP11-40C6.2. (F) IF staining assay was performed to detect YAP1 nuclear translocation in SMMC-7721 and Hep3B cells transfected with WT and MU RP11-40C6.2. (G) Transwell cell migration assay was used to assess invasion of SMMC-7721 and Hep3B cells transfected with WT and MU RP11-40C6.2. Mean±SEM values from at least three independent experiments are presented. * p <0.05, ** p <0.01.

    Techniques Used: Binding Assay, Western Blot, Transfection, Staining, Translocation Assay, Cell Migration Assay

    polyclonal rabbit abs  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc polyclonal rabbit abs
    Generation of chimeric human/mouse CD79 knockin mice. (A) Schematic representation of B cell Ag receptor complex. (B) CD79 amino acid conservation between mice and humans. Black intervals represent regions of 0% amino acid conservation. Domain demarcation: CY, cytoplasmic; EC, extracellular; L, leader; TM, transmembrane. Conserved cysteines required for interchain disulfide bonding shown for each. (C) Schematic representation of cCD79. Human sequences comprise the CD79B extracellular domain and CD79A extracellular/transmembrane domains. (D) Surface staining of splenic B cells (B220+) from chimeric (h/m)CD79 knockin mice and control C57BL/6 mice. Gray lines show B220−. (E) Rabbit anti-mCD79 immunoblot analysis of whole-cell lysates (purified splenic B cells, CD43−) from cCD79 and WT mice. Upper membrane probed with a <t>polyclonal</t> rabbit Ab raised against a mCD79A and mCD79B extracellular domain fusion protein (Cambier laboratory). Middle membrane probed with a polyclonal rabbit Ab raised against the cytoplasmic domain of mCD79B (Cambier laboratory). An anti–β actin blot was used as a protein loading control. (F) Surface staining of B cell gated (CD19+) human PBMCs with anti-hCD79B (AT-105). Gray line shows CD19−. (G) Surface staining as a function of cCD79B allele dosage. Splenocytes from cCD79 mice of the indicated genotypes were stained with both anti-hCD79B (AT-105) and anti-mCD79B (HM79). Gray contour shows B220−. (H) IgM and IgD surface expression in chimeric mice described in (G). Gray line shows B220−. n = 4 female mice per group for (G) and (H). Error bars represent SEM. All data represent at least three independent experiments; representative data are shown.
    Polyclonal Rabbit Abs, 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/polyclonal rabbit abs/product/Cell Signaling Technology Inc
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    polyclonal rabbit abs - by Bioz Stars, 2023-03
    86/100 stars

    Images

    1) Product Images from "Preclinical analysis of candidate anti-human CD79 therapeutic antibodies using a humanized CD79 mouse model"

    Article Title: Preclinical analysis of candidate anti-human CD79 therapeutic antibodies using a humanized CD79 mouse model

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.2101056

    Generation of chimeric human/mouse CD79 knockin mice. (A) Schematic representation of B cell Ag receptor complex. (B) CD79 amino acid conservation between mice and humans. Black intervals represent regions of 0% amino acid conservation. Domain demarcation: CY, cytoplasmic; EC, extracellular; L, leader; TM, transmembrane. Conserved cysteines required for interchain disulfide bonding shown for each. (C) Schematic representation of cCD79. Human sequences comprise the CD79B extracellular domain and CD79A extracellular/transmembrane domains. (D) Surface staining of splenic B cells (B220+) from chimeric (h/m)CD79 knockin mice and control C57BL/6 mice. Gray lines show B220−. (E) Rabbit anti-mCD79 immunoblot analysis of whole-cell lysates (purified splenic B cells, CD43−) from cCD79 and WT mice. Upper membrane probed with a polyclonal rabbit Ab raised against a mCD79A and mCD79B extracellular domain fusion protein (Cambier laboratory). Middle membrane probed with a polyclonal rabbit Ab raised against the cytoplasmic domain of mCD79B (Cambier laboratory). An anti–β actin blot was used as a protein loading control. (F) Surface staining of B cell gated (CD19+) human PBMCs with anti-hCD79B (AT-105). Gray line shows CD19−. (G) Surface staining as a function of cCD79B allele dosage. Splenocytes from cCD79 mice of the indicated genotypes were stained with both anti-hCD79B (AT-105) and anti-mCD79B (HM79). Gray contour shows B220−. (H) IgM and IgD surface expression in chimeric mice described in (G). Gray line shows B220−. n = 4 female mice per group for (G) and (H). Error bars represent SEM. All data represent at least three independent experiments; representative data are shown.
    Figure Legend Snippet: Generation of chimeric human/mouse CD79 knockin mice. (A) Schematic representation of B cell Ag receptor complex. (B) CD79 amino acid conservation between mice and humans. Black intervals represent regions of 0% amino acid conservation. Domain demarcation: CY, cytoplasmic; EC, extracellular; L, leader; TM, transmembrane. Conserved cysteines required for interchain disulfide bonding shown for each. (C) Schematic representation of cCD79. Human sequences comprise the CD79B extracellular domain and CD79A extracellular/transmembrane domains. (D) Surface staining of splenic B cells (B220+) from chimeric (h/m)CD79 knockin mice and control C57BL/6 mice. Gray lines show B220−. (E) Rabbit anti-mCD79 immunoblot analysis of whole-cell lysates (purified splenic B cells, CD43−) from cCD79 and WT mice. Upper membrane probed with a polyclonal rabbit Ab raised against a mCD79A and mCD79B extracellular domain fusion protein (Cambier laboratory). Middle membrane probed with a polyclonal rabbit Ab raised against the cytoplasmic domain of mCD79B (Cambier laboratory). An anti–β actin blot was used as a protein loading control. (F) Surface staining of B cell gated (CD19+) human PBMCs with anti-hCD79B (AT-105). Gray line shows CD19−. (G) Surface staining as a function of cCD79B allele dosage. Splenocytes from cCD79 mice of the indicated genotypes were stained with both anti-hCD79B (AT-105) and anti-mCD79B (HM79). Gray contour shows B220−. (H) IgM and IgD surface expression in chimeric mice described in (G). Gray line shows B220−. n = 4 female mice per group for (G) and (H). Error bars represent SEM. All data represent at least three independent experiments; representative data are shown.

    Techniques Used: Knock-In, Staining, Western Blot, Purification, Expressing

    BCR signaling and B cell immune responses are unaffected by expression of cCD79. (A) BCR-mediated global tyrosine phosphorylation in chimeric and control B cells. Cell equivalents (2 × 106; CD43−) per lane, resting or stimulated with 10 µg/ml rabbit F(ab′)2 anti-mIg (H+L) for 5 min. Unstimulated cells were run in parallel (left four lanes) to show basal phosphorylation levels. Protein-laden PVDF membranes probed with Abs against p-Tyr (4G10) and actin. (B) BCR-mediated CD79A phosphorylation. Prepared as in (A), probed with anti–p-CD79A (Y182) (rabbit polyclonal anti-mouse pCD79A [Y182]). An anti-mCD79B (see (Fig. 1E), which recognizes the cytoplasmic tail of mCD79B, was used together with actin to normalize the relative abundance of phosphorylated mCD79A (p-CD79A/p-CD79B/actin). Normalized band densities are depicted to the right. (C) BCR-mediated Syk phosphorylation. Prepared as in (A), probed with anti–p-Syk (Y252) (polyclonal rabbit anti–p-Syk [Y525/526]) and biotinylated anti-Syk (in-house) followed by fluorescently conjugated streptavidin. Relative, normalized band densities (pSyk/Syk) are depicted to the right. (D) Representative relative intracellular free calcium before and after BCR stimulation. Splenocytes, stained with anti-B220 and loaded with Indo-1 AM, were stimulated with 1 or 10 µg/ml F(ab′)2 of either goat anti-mIgM (upper traces) or rabbit anti-mIg (H+L) (lower), approximating IgM-only and total BCR stimulation, respectively. Poststimulation, basal-normalized area under the curve (AUC) is depicted on the right. (E) Total (NP19-binding) and high affinity (NP2-binding) IgG+ ELISPOT quantification, 16 d postimmunization with NP-conjugated OVA in alum. (F) Relative serum concentrations of IgG anti-NP Abs from mice immunized in (E) at 16 d postimmunization. NI, not immunized. (G) Total (NP19 binding) IgM+ ELISPOT quantification, at 7 days postimmunization with NP59-Ficoll. n = 3 male and 3 female mice per group. Error bars show SEM. All data represent at least three independent experiments; representative data are shown.
    Figure Legend Snippet: BCR signaling and B cell immune responses are unaffected by expression of cCD79. (A) BCR-mediated global tyrosine phosphorylation in chimeric and control B cells. Cell equivalents (2 × 106; CD43−) per lane, resting or stimulated with 10 µg/ml rabbit F(ab′)2 anti-mIg (H+L) for 5 min. Unstimulated cells were run in parallel (left four lanes) to show basal phosphorylation levels. Protein-laden PVDF membranes probed with Abs against p-Tyr (4G10) and actin. (B) BCR-mediated CD79A phosphorylation. Prepared as in (A), probed with anti–p-CD79A (Y182) (rabbit polyclonal anti-mouse pCD79A [Y182]). An anti-mCD79B (see (Fig. 1E), which recognizes the cytoplasmic tail of mCD79B, was used together with actin to normalize the relative abundance of phosphorylated mCD79A (p-CD79A/p-CD79B/actin). Normalized band densities are depicted to the right. (C) BCR-mediated Syk phosphorylation. Prepared as in (A), probed with anti–p-Syk (Y252) (polyclonal rabbit anti–p-Syk [Y525/526]) and biotinylated anti-Syk (in-house) followed by fluorescently conjugated streptavidin. Relative, normalized band densities (pSyk/Syk) are depicted to the right. (D) Representative relative intracellular free calcium before and after BCR stimulation. Splenocytes, stained with anti-B220 and loaded with Indo-1 AM, were stimulated with 1 or 10 µg/ml F(ab′)2 of either goat anti-mIgM (upper traces) or rabbit anti-mIg (H+L) (lower), approximating IgM-only and total BCR stimulation, respectively. Poststimulation, basal-normalized area under the curve (AUC) is depicted on the right. (E) Total (NP19-binding) and high affinity (NP2-binding) IgG+ ELISPOT quantification, 16 d postimmunization with NP-conjugated OVA in alum. (F) Relative serum concentrations of IgG anti-NP Abs from mice immunized in (E) at 16 d postimmunization. NI, not immunized. (G) Total (NP19 binding) IgM+ ELISPOT quantification, at 7 days postimmunization with NP59-Ficoll. n = 3 male and 3 female mice per group. Error bars show SEM. All data represent at least three independent experiments; representative data are shown.

    Techniques Used: Expressing, Staining, Binding Assay, Enzyme-linked Immunospot

    Anti-hCD79A treatment induces suppression of BCR-mediated calcium mobilization. (A) BCR-mediated calcium signaling in B cells from cCD79A mice receiving a 250-µg i.p. injection of anti-hCD79A D265A or control hIgG4 24 h prior. mBCR expression (left) was measured and gated by staining with a polyclonal goat Fab anti-mIgG (H+L). Anti-hCD79A, solid line; control hIgG4, dashed line. Cells were restimulated with 1 or 10 µg/ml rat anti-mIgM (B76). The poststimulation area under the curve (AUC) was normalized to basal calcium levels (right). (B) Cells prepared as in (A) stimulated with 10 µM ionomycin. (C) BCR-mediated calcium signaling in Ramos cells after overnight in vitro incubation with 25 µg/ml hIgG4 anti-hCD79A D265A (solid line) or isotype control hIgG4 (dashed line). Cells were restimulated with either 1 or 10 µg/ml goat F(ab′)2 anti-hIgM Cµ5. (D) Flow cytometric (left and mean fluorescence intensity [MFI] bar graph) and Western blot (right and densitometry) analysis of PTEN expression in B cells from cCD79A animals receiving a 250-µg i.p. injection of either anti-hCD79A D265A or control hIgG4, 24 h prior. For flow cytometry, RBC-lysed splenocytes were fixed and permeabilized before staining with Abs against B220 and PTEN. Gray histogram represents staining isotype control Ab. hIgG4, dashed line; anti-hCD79A D265A, solid line. For Western blot, membranes were prepared as in (Fig. 3D, without BCR stimulation, before being probed with Abs against PTEN or actin. (E) hCD20-CreTAM × ROSA26-STOPflox-YFP × PTENflox/flox or hCD20-CreTAM × ROSA-26-STOPflox-YFP × PTENWT mice were given 2-mg i.p. injections of tamoxifen (TAM) on day 0. On day 7 after TAM, mice were injected i.p. with 0.25 mg of either mIgG2a D265A anti-mCD79B or control mIgG2a anti-HEL. Eighteen hours after Ab injection, B220+YFP+ splenocytes were analyzed by flow for PTEN expression and Ab coating. (F) As before, calcium was measured as a function of equal mBCR expression. n = 3 mice per group. Error bars show SEM. All data represents at least three independent experiments; representative data are shown. A Student t test was used to evaluate statistical significance. *p < 0.05, **p < 0.01, ****p < 0.0001.
    Figure Legend Snippet: Anti-hCD79A treatment induces suppression of BCR-mediated calcium mobilization. (A) BCR-mediated calcium signaling in B cells from cCD79A mice receiving a 250-µg i.p. injection of anti-hCD79A D265A or control hIgG4 24 h prior. mBCR expression (left) was measured and gated by staining with a polyclonal goat Fab anti-mIgG (H+L). Anti-hCD79A, solid line; control hIgG4, dashed line. Cells were restimulated with 1 or 10 µg/ml rat anti-mIgM (B76). The poststimulation area under the curve (AUC) was normalized to basal calcium levels (right). (B) Cells prepared as in (A) stimulated with 10 µM ionomycin. (C) BCR-mediated calcium signaling in Ramos cells after overnight in vitro incubation with 25 µg/ml hIgG4 anti-hCD79A D265A (solid line) or isotype control hIgG4 (dashed line). Cells were restimulated with either 1 or 10 µg/ml goat F(ab′)2 anti-hIgM Cµ5. (D) Flow cytometric (left and mean fluorescence intensity [MFI] bar graph) and Western blot (right and densitometry) analysis of PTEN expression in B cells from cCD79A animals receiving a 250-µg i.p. injection of either anti-hCD79A D265A or control hIgG4, 24 h prior. For flow cytometry, RBC-lysed splenocytes were fixed and permeabilized before staining with Abs against B220 and PTEN. Gray histogram represents staining isotype control Ab. hIgG4, dashed line; anti-hCD79A D265A, solid line. For Western blot, membranes were prepared as in (Fig. 3D, without BCR stimulation, before being probed with Abs against PTEN or actin. (E) hCD20-CreTAM × ROSA26-STOPflox-YFP × PTENflox/flox or hCD20-CreTAM × ROSA-26-STOPflox-YFP × PTENWT mice were given 2-mg i.p. injections of tamoxifen (TAM) on day 0. On day 7 after TAM, mice were injected i.p. with 0.25 mg of either mIgG2a D265A anti-mCD79B or control mIgG2a anti-HEL. Eighteen hours after Ab injection, B220+YFP+ splenocytes were analyzed by flow for PTEN expression and Ab coating. (F) As before, calcium was measured as a function of equal mBCR expression. n = 3 mice per group. Error bars show SEM. All data represents at least three independent experiments; representative data are shown. A Student t test was used to evaluate statistical significance. *p < 0.05, **p < 0.01, ****p < 0.0001.

    Techniques Used: Injection, Expressing, Staining, In Vitro, Incubation, Fluorescence, Western Blot, Flow Cytometry

    polyclonal rabbit abs  (Cell Signaling Technology Inc)


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    Structured Review

    Cell Signaling Technology Inc polyclonal rabbit abs
    Generation of chimeric human/mouse CD79 knockin mice. (A) Schematic representation of B cell Ag receptor complex. (B) CD79 amino acid conservation between mice and humans. Black intervals represent regions of 0% amino acid conservation. Domain demarcation: CY, cytoplasmic; EC, extracellular; L, leader; TM, transmembrane. Conserved cysteines required for interchain disulfide bonding shown for each. (C) Schematic representation of cCD79. Human sequences comprise the CD79B extracellular domain and CD79A extracellular/transmembrane domains. (D) Surface staining of splenic B cells (B220+) from chimeric (h/m)CD79 knockin mice and control C57BL/6 mice. Gray lines show B220−. (E) Rabbit anti-mCD79 immunoblot analysis of whole-cell lysates (purified splenic B cells, CD43−) from cCD79 and WT mice. Upper membrane probed with a <t>polyclonal</t> rabbit Ab raised against a mCD79A and mCD79B extracellular domain fusion protein (Cambier laboratory). Middle membrane probed with a polyclonal rabbit Ab raised against the cytoplasmic domain of mCD79B (Cambier laboratory). An anti–β actin blot was used as a protein loading control. (F) Surface staining of B cell gated (CD19+) human PBMCs with anti-hCD79B (AT-105). Gray line shows CD19−. (G) Surface staining as a function of cCD79B allele dosage. Splenocytes from cCD79 mice of the indicated genotypes were stained with both anti-hCD79B (AT-105) and anti-mCD79B (HM79). Gray contour shows B220−. (H) IgM and IgD surface expression in chimeric mice described in (G). Gray line shows B220−. n = 4 female mice per group for (G) and (H). Error bars represent SEM. All data represent at least three independent experiments; representative data are shown.
    Polyclonal Rabbit Abs, 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/polyclonal rabbit abs/product/Cell Signaling Technology Inc
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    polyclonal rabbit abs - by Bioz Stars, 2023-03
    86/100 stars

    Images

    1) Product Images from "Preclinical analysis of candidate anti-human CD79 therapeutic antibodies using a humanized CD79 mouse model"

    Article Title: Preclinical analysis of candidate anti-human CD79 therapeutic antibodies using a humanized CD79 mouse model

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.2101056

    Generation of chimeric human/mouse CD79 knockin mice. (A) Schematic representation of B cell Ag receptor complex. (B) CD79 amino acid conservation between mice and humans. Black intervals represent regions of 0% amino acid conservation. Domain demarcation: CY, cytoplasmic; EC, extracellular; L, leader; TM, transmembrane. Conserved cysteines required for interchain disulfide bonding shown for each. (C) Schematic representation of cCD79. Human sequences comprise the CD79B extracellular domain and CD79A extracellular/transmembrane domains. (D) Surface staining of splenic B cells (B220+) from chimeric (h/m)CD79 knockin mice and control C57BL/6 mice. Gray lines show B220−. (E) Rabbit anti-mCD79 immunoblot analysis of whole-cell lysates (purified splenic B cells, CD43−) from cCD79 and WT mice. Upper membrane probed with a polyclonal rabbit Ab raised against a mCD79A and mCD79B extracellular domain fusion protein (Cambier laboratory). Middle membrane probed with a polyclonal rabbit Ab raised against the cytoplasmic domain of mCD79B (Cambier laboratory). An anti–β actin blot was used as a protein loading control. (F) Surface staining of B cell gated (CD19+) human PBMCs with anti-hCD79B (AT-105). Gray line shows CD19−. (G) Surface staining as a function of cCD79B allele dosage. Splenocytes from cCD79 mice of the indicated genotypes were stained with both anti-hCD79B (AT-105) and anti-mCD79B (HM79). Gray contour shows B220−. (H) IgM and IgD surface expression in chimeric mice described in (G). Gray line shows B220−. n = 4 female mice per group for (G) and (H). Error bars represent SEM. All data represent at least three independent experiments; representative data are shown.
    Figure Legend Snippet: Generation of chimeric human/mouse CD79 knockin mice. (A) Schematic representation of B cell Ag receptor complex. (B) CD79 amino acid conservation between mice and humans. Black intervals represent regions of 0% amino acid conservation. Domain demarcation: CY, cytoplasmic; EC, extracellular; L, leader; TM, transmembrane. Conserved cysteines required for interchain disulfide bonding shown for each. (C) Schematic representation of cCD79. Human sequences comprise the CD79B extracellular domain and CD79A extracellular/transmembrane domains. (D) Surface staining of splenic B cells (B220+) from chimeric (h/m)CD79 knockin mice and control C57BL/6 mice. Gray lines show B220−. (E) Rabbit anti-mCD79 immunoblot analysis of whole-cell lysates (purified splenic B cells, CD43−) from cCD79 and WT mice. Upper membrane probed with a polyclonal rabbit Ab raised against a mCD79A and mCD79B extracellular domain fusion protein (Cambier laboratory). Middle membrane probed with a polyclonal rabbit Ab raised against the cytoplasmic domain of mCD79B (Cambier laboratory). An anti–β actin blot was used as a protein loading control. (F) Surface staining of B cell gated (CD19+) human PBMCs with anti-hCD79B (AT-105). Gray line shows CD19−. (G) Surface staining as a function of cCD79B allele dosage. Splenocytes from cCD79 mice of the indicated genotypes were stained with both anti-hCD79B (AT-105) and anti-mCD79B (HM79). Gray contour shows B220−. (H) IgM and IgD surface expression in chimeric mice described in (G). Gray line shows B220−. n = 4 female mice per group for (G) and (H). Error bars represent SEM. All data represent at least three independent experiments; representative data are shown.

    Techniques Used: Knock-In, Staining, Western Blot, Purification, Expressing

    BCR signaling and B cell immune responses are unaffected by expression of cCD79. (A) BCR-mediated global tyrosine phosphorylation in chimeric and control B cells. Cell equivalents (2 × 106; CD43−) per lane, resting or stimulated with 10 µg/ml rabbit F(ab′)2 anti-mIg (H+L) for 5 min. Unstimulated cells were run in parallel (left four lanes) to show basal phosphorylation levels. Protein-laden PVDF membranes probed with Abs against p-Tyr (4G10) and actin. (B) BCR-mediated CD79A phosphorylation. Prepared as in (A), probed with anti–p-CD79A (Y182) (rabbit polyclonal anti-mouse pCD79A [Y182]). An anti-mCD79B (see (Fig. 1E), which recognizes the cytoplasmic tail of mCD79B, was used together with actin to normalize the relative abundance of phosphorylated mCD79A (p-CD79A/p-CD79B/actin). Normalized band densities are depicted to the right. (C) BCR-mediated Syk phosphorylation. Prepared as in (A), probed with anti–p-Syk (Y252) (polyclonal rabbit anti–p-Syk [Y525/526]) and biotinylated anti-Syk (in-house) followed by fluorescently conjugated streptavidin. Relative, normalized band densities (pSyk/Syk) are depicted to the right. (D) Representative relative intracellular free calcium before and after BCR stimulation. Splenocytes, stained with anti-B220 and loaded with Indo-1 AM, were stimulated with 1 or 10 µg/ml F(ab′)2 of either goat anti-mIgM (upper traces) or rabbit anti-mIg (H+L) (lower), approximating IgM-only and total BCR stimulation, respectively. Poststimulation, basal-normalized area under the curve (AUC) is depicted on the right. (E) Total (NP19-binding) and high affinity (NP2-binding) IgG+ ELISPOT quantification, 16 d postimmunization with NP-conjugated OVA in alum. (F) Relative serum concentrations of IgG anti-NP Abs from mice immunized in (E) at 16 d postimmunization. NI, not immunized. (G) Total (NP19 binding) IgM+ ELISPOT quantification, at 7 days postimmunization with NP59-Ficoll. n = 3 male and 3 female mice per group. Error bars show SEM. All data represent at least three independent experiments; representative data are shown.
    Figure Legend Snippet: BCR signaling and B cell immune responses are unaffected by expression of cCD79. (A) BCR-mediated global tyrosine phosphorylation in chimeric and control B cells. Cell equivalents (2 × 106; CD43−) per lane, resting or stimulated with 10 µg/ml rabbit F(ab′)2 anti-mIg (H+L) for 5 min. Unstimulated cells were run in parallel (left four lanes) to show basal phosphorylation levels. Protein-laden PVDF membranes probed with Abs against p-Tyr (4G10) and actin. (B) BCR-mediated CD79A phosphorylation. Prepared as in (A), probed with anti–p-CD79A (Y182) (rabbit polyclonal anti-mouse pCD79A [Y182]). An anti-mCD79B (see (Fig. 1E), which recognizes the cytoplasmic tail of mCD79B, was used together with actin to normalize the relative abundance of phosphorylated mCD79A (p-CD79A/p-CD79B/actin). Normalized band densities are depicted to the right. (C) BCR-mediated Syk phosphorylation. Prepared as in (A), probed with anti–p-Syk (Y252) (polyclonal rabbit anti–p-Syk [Y525/526]) and biotinylated anti-Syk (in-house) followed by fluorescently conjugated streptavidin. Relative, normalized band densities (pSyk/Syk) are depicted to the right. (D) Representative relative intracellular free calcium before and after BCR stimulation. Splenocytes, stained with anti-B220 and loaded with Indo-1 AM, were stimulated with 1 or 10 µg/ml F(ab′)2 of either goat anti-mIgM (upper traces) or rabbit anti-mIg (H+L) (lower), approximating IgM-only and total BCR stimulation, respectively. Poststimulation, basal-normalized area under the curve (AUC) is depicted on the right. (E) Total (NP19-binding) and high affinity (NP2-binding) IgG+ ELISPOT quantification, 16 d postimmunization with NP-conjugated OVA in alum. (F) Relative serum concentrations of IgG anti-NP Abs from mice immunized in (E) at 16 d postimmunization. NI, not immunized. (G) Total (NP19 binding) IgM+ ELISPOT quantification, at 7 days postimmunization with NP59-Ficoll. n = 3 male and 3 female mice per group. Error bars show SEM. All data represent at least three independent experiments; representative data are shown.

    Techniques Used: Expressing, Staining, Binding Assay, Enzyme-linked Immunospot

    Anti-hCD79A treatment induces suppression of BCR-mediated calcium mobilization. (A) BCR-mediated calcium signaling in B cells from cCD79A mice receiving a 250-µg i.p. injection of anti-hCD79A D265A or control hIgG4 24 h prior. mBCR expression (left) was measured and gated by staining with a polyclonal goat Fab anti-mIgG (H+L). Anti-hCD79A, solid line; control hIgG4, dashed line. Cells were restimulated with 1 or 10 µg/ml rat anti-mIgM (B76). The poststimulation area under the curve (AUC) was normalized to basal calcium levels (right). (B) Cells prepared as in (A) stimulated with 10 µM ionomycin. (C) BCR-mediated calcium signaling in Ramos cells after overnight in vitro incubation with 25 µg/ml hIgG4 anti-hCD79A D265A (solid line) or isotype control hIgG4 (dashed line). Cells were restimulated with either 1 or 10 µg/ml goat F(ab′)2 anti-hIgM Cµ5. (D) Flow cytometric (left and mean fluorescence intensity [MFI] bar graph) and Western blot (right and densitometry) analysis of PTEN expression in B cells from cCD79A animals receiving a 250-µg i.p. injection of either anti-hCD79A D265A or control hIgG4, 24 h prior. For flow cytometry, RBC-lysed splenocytes were fixed and permeabilized before staining with Abs against B220 and PTEN. Gray histogram represents staining isotype control Ab. hIgG4, dashed line; anti-hCD79A D265A, solid line. For Western blot, membranes were prepared as in (Fig. 3D, without BCR stimulation, before being probed with Abs against PTEN or actin. (E) hCD20-CreTAM × ROSA26-STOPflox-YFP × PTENflox/flox or hCD20-CreTAM × ROSA-26-STOPflox-YFP × PTENWT mice were given 2-mg i.p. injections of tamoxifen (TAM) on day 0. On day 7 after TAM, mice were injected i.p. with 0.25 mg of either mIgG2a D265A anti-mCD79B or control mIgG2a anti-HEL. Eighteen hours after Ab injection, B220+YFP+ splenocytes were analyzed by flow for PTEN expression and Ab coating. (F) As before, calcium was measured as a function of equal mBCR expression. n = 3 mice per group. Error bars show SEM. All data represents at least three independent experiments; representative data are shown. A Student t test was used to evaluate statistical significance. *p < 0.05, **p < 0.01, ****p < 0.0001.
    Figure Legend Snippet: Anti-hCD79A treatment induces suppression of BCR-mediated calcium mobilization. (A) BCR-mediated calcium signaling in B cells from cCD79A mice receiving a 250-µg i.p. injection of anti-hCD79A D265A or control hIgG4 24 h prior. mBCR expression (left) was measured and gated by staining with a polyclonal goat Fab anti-mIgG (H+L). Anti-hCD79A, solid line; control hIgG4, dashed line. Cells were restimulated with 1 or 10 µg/ml rat anti-mIgM (B76). The poststimulation area under the curve (AUC) was normalized to basal calcium levels (right). (B) Cells prepared as in (A) stimulated with 10 µM ionomycin. (C) BCR-mediated calcium signaling in Ramos cells after overnight in vitro incubation with 25 µg/ml hIgG4 anti-hCD79A D265A (solid line) or isotype control hIgG4 (dashed line). Cells were restimulated with either 1 or 10 µg/ml goat F(ab′)2 anti-hIgM Cµ5. (D) Flow cytometric (left and mean fluorescence intensity [MFI] bar graph) and Western blot (right and densitometry) analysis of PTEN expression in B cells from cCD79A animals receiving a 250-µg i.p. injection of either anti-hCD79A D265A or control hIgG4, 24 h prior. For flow cytometry, RBC-lysed splenocytes were fixed and permeabilized before staining with Abs against B220 and PTEN. Gray histogram represents staining isotype control Ab. hIgG4, dashed line; anti-hCD79A D265A, solid line. For Western blot, membranes were prepared as in (Fig. 3D, without BCR stimulation, before being probed with Abs against PTEN or actin. (E) hCD20-CreTAM × ROSA26-STOPflox-YFP × PTENflox/flox or hCD20-CreTAM × ROSA-26-STOPflox-YFP × PTENWT mice were given 2-mg i.p. injections of tamoxifen (TAM) on day 0. On day 7 after TAM, mice were injected i.p. with 0.25 mg of either mIgG2a D265A anti-mCD79B or control mIgG2a anti-HEL. Eighteen hours after Ab injection, B220+YFP+ splenocytes were analyzed by flow for PTEN expression and Ab coating. (F) As before, calcium was measured as a function of equal mBCR expression. n = 3 mice per group. Error bars show SEM. All data represents at least three independent experiments; representative data are shown. A Student t test was used to evaluate statistical significance. *p < 0.05, **p < 0.01, ****p < 0.0001.

    Techniques Used: Injection, Expressing, Staining, In Vitro, Incubation, Fluorescence, Western Blot, Flow Cytometry

    mouse anti β actin polyclonal antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc mouse anti β actin polyclonal antibody
    Mouse Anti β Actin Polyclonal Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    rabbit polyclonal phospho tacc3 antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit polyclonal phospho tacc3 antibody
    ( A ) Schematic representation of <t>GFP-TACC3</t> (Δ678–681)-TACC3 shRNA and GFP-TACC3 (Δ682–688)-TACC3 shRNA constructs. ( B ) Lysates of HeLa Kyoto cells transfected with GFP-TACC3 (WT)-TACC3 shRNA, GFP-TACC3 (Δ682–688)-TACC3 shRNA, or GFP-TACC3 (Δ678–681)-TACC3 shRNA for 48 h were analyzed for exogenous TACC3 proteins with simultaneous depletion of endogenous TACC3. Both endogenous TACC3 and GFP-TACC3 proteins were stained with mouse monoclonal TACC3 antibody. Actin was probed as a control. TACC3 level in control untrasfected cells is also shown. ( C ) Immunoprecipitation of GFP-tagged TACC3 proteins using the anti-GFP antibody in the lysates of HeLa Kyoto cells transfected with GFP-TACC3 (WT)-TACC3 shRNA, GFP-TACC3 (Δ678–681)-TACC3 shRNA, or GFP-TACC3 (Δ682–688)-TACC3 shRNA. The immunoblots were probed with GFP and ch-TOG antibodies. ( D ) Representative confocal images of GFP-TACC3 (WT)-TACC3 shRNA (top panel), GFP-TACC3 (Δ678-681)-TACC3 shRNA (middle panel), or GFP-TACC3 (Δ682–688)-TACC3 shRNA (bottom panel)- transfected mitotic HeLa Kyoto cells showing the differences in γ-tubulin levels at the centrosomes. Scale bar, 5 μm, γ-tubulin, and ch-TOG were stained with mouse monoclonal anti-γ-tubulin and rabbit polyclonal anti-ch-TOG antibody, respectively. ( E ) The bar graph shows the quantification of γ-tubulin intensity at the centrosomes (two) in different conditions as of (d). ( F ) Representative confocal images of GFP-TACC3 (WT)-TACC3 shRNA (top panel), GFP-TACC3 (Δ678–681)-TACC3 shRNA (middle panel), or GFP-TACC3 (Δ682–688)-TACC3 shRNA (bottom panel) transfected mitotic HeLa Kyoto cells showing the differences in GCP6 localization at centrosomes. Scale bar, 5 μm, GCP6 was stained with mouse monoclonal anti-GCP6 antibody. ( G ) The bar graph shows the quantification of GCP6 intensity at the centrosomes in different conditions as in panel (F). ( H ) The bar graph shows the percentage of cells (cells with above the average centrosomal γ-tubulin intensity of TACC3 WT-expressed cells) with increased γ-tubulin intensity phenotype in both the mutant cases. The bars in (e), (g), and (h) represent mean ± S.E. The number of mitotic cells counted = 100 each (four independent experiments); ****, P <0.0001, ***, P <0.001.
    Rabbit Polyclonal Phospho Tacc3 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
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    1) Product Images from "TACC3–ch-TOG interaction regulates spindle microtubule assembly by controlling centrosomal recruitment of γ-TuRC"

    Article Title: TACC3–ch-TOG interaction regulates spindle microtubule assembly by controlling centrosomal recruitment of γ-TuRC

    Journal: Bioscience Reports

    doi: 10.1042/BSR20221882

    ( A ) Schematic representation of GFP-TACC3 (Δ678–681)-TACC3 shRNA and GFP-TACC3 (Δ682–688)-TACC3 shRNA constructs. ( B ) Lysates of HeLa Kyoto cells transfected with GFP-TACC3 (WT)-TACC3 shRNA, GFP-TACC3 (Δ682–688)-TACC3 shRNA, or GFP-TACC3 (Δ678–681)-TACC3 shRNA for 48 h were analyzed for exogenous TACC3 proteins with simultaneous depletion of endogenous TACC3. Both endogenous TACC3 and GFP-TACC3 proteins were stained with mouse monoclonal TACC3 antibody. Actin was probed as a control. TACC3 level in control untrasfected cells is also shown. ( C ) Immunoprecipitation of GFP-tagged TACC3 proteins using the anti-GFP antibody in the lysates of HeLa Kyoto cells transfected with GFP-TACC3 (WT)-TACC3 shRNA, GFP-TACC3 (Δ678–681)-TACC3 shRNA, or GFP-TACC3 (Δ682–688)-TACC3 shRNA. The immunoblots were probed with GFP and ch-TOG antibodies. ( D ) Representative confocal images of GFP-TACC3 (WT)-TACC3 shRNA (top panel), GFP-TACC3 (Δ678-681)-TACC3 shRNA (middle panel), or GFP-TACC3 (Δ682–688)-TACC3 shRNA (bottom panel)- transfected mitotic HeLa Kyoto cells showing the differences in γ-tubulin levels at the centrosomes. Scale bar, 5 μm, γ-tubulin, and ch-TOG were stained with mouse monoclonal anti-γ-tubulin and rabbit polyclonal anti-ch-TOG antibody, respectively. ( E ) The bar graph shows the quantification of γ-tubulin intensity at the centrosomes (two) in different conditions as of (d). ( F ) Representative confocal images of GFP-TACC3 (WT)-TACC3 shRNA (top panel), GFP-TACC3 (Δ678–681)-TACC3 shRNA (middle panel), or GFP-TACC3 (Δ682–688)-TACC3 shRNA (bottom panel) transfected mitotic HeLa Kyoto cells showing the differences in GCP6 localization at centrosomes. Scale bar, 5 μm, GCP6 was stained with mouse monoclonal anti-GCP6 antibody. ( G ) The bar graph shows the quantification of GCP6 intensity at the centrosomes in different conditions as in panel (F). ( H ) The bar graph shows the percentage of cells (cells with above the average centrosomal γ-tubulin intensity of TACC3 WT-expressed cells) with increased γ-tubulin intensity phenotype in both the mutant cases. The bars in (e), (g), and (h) represent mean ± S.E. The number of mitotic cells counted = 100 each (four independent experiments); ****, P <0.0001, ***, P <0.001.
    Figure Legend Snippet: ( A ) Schematic representation of GFP-TACC3 (Δ678–681)-TACC3 shRNA and GFP-TACC3 (Δ682–688)-TACC3 shRNA constructs. ( B ) Lysates of HeLa Kyoto cells transfected with GFP-TACC3 (WT)-TACC3 shRNA, GFP-TACC3 (Δ682–688)-TACC3 shRNA, or GFP-TACC3 (Δ678–681)-TACC3 shRNA for 48 h were analyzed for exogenous TACC3 proteins with simultaneous depletion of endogenous TACC3. Both endogenous TACC3 and GFP-TACC3 proteins were stained with mouse monoclonal TACC3 antibody. Actin was probed as a control. TACC3 level in control untrasfected cells is also shown. ( C ) Immunoprecipitation of GFP-tagged TACC3 proteins using the anti-GFP antibody in the lysates of HeLa Kyoto cells transfected with GFP-TACC3 (WT)-TACC3 shRNA, GFP-TACC3 (Δ678–681)-TACC3 shRNA, or GFP-TACC3 (Δ682–688)-TACC3 shRNA. The immunoblots were probed with GFP and ch-TOG antibodies. ( D ) Representative confocal images of GFP-TACC3 (WT)-TACC3 shRNA (top panel), GFP-TACC3 (Δ678-681)-TACC3 shRNA (middle panel), or GFP-TACC3 (Δ682–688)-TACC3 shRNA (bottom panel)- transfected mitotic HeLa Kyoto cells showing the differences in γ-tubulin levels at the centrosomes. Scale bar, 5 μm, γ-tubulin, and ch-TOG were stained with mouse monoclonal anti-γ-tubulin and rabbit polyclonal anti-ch-TOG antibody, respectively. ( E ) The bar graph shows the quantification of γ-tubulin intensity at the centrosomes (two) in different conditions as of (d). ( F ) Representative confocal images of GFP-TACC3 (WT)-TACC3 shRNA (top panel), GFP-TACC3 (Δ678–681)-TACC3 shRNA (middle panel), or GFP-TACC3 (Δ682–688)-TACC3 shRNA (bottom panel) transfected mitotic HeLa Kyoto cells showing the differences in GCP6 localization at centrosomes. Scale bar, 5 μm, GCP6 was stained with mouse monoclonal anti-GCP6 antibody. ( G ) The bar graph shows the quantification of GCP6 intensity at the centrosomes in different conditions as in panel (F). ( H ) The bar graph shows the percentage of cells (cells with above the average centrosomal γ-tubulin intensity of TACC3 WT-expressed cells) with increased γ-tubulin intensity phenotype in both the mutant cases. The bars in (e), (g), and (h) represent mean ± S.E. The number of mitotic cells counted = 100 each (four independent experiments); ****, P <0.0001, ***, P <0.001.

    Techniques Used: shRNA, Construct, Transfection, Staining, Immunoprecipitation, Western Blot, Mutagenesis

    ( A ) Representative confocal images of GFP-TACC3 (WT)-TACC3 shRNA (top panel), GFP-TACC3 (Δ678–681)-TACC3 shRNA (middle panel), or GFP-TACC3 (Δ682–688)-TACC3 shRNA (bottom panel) transfected mitotic HeLa Kyoto cells showing the differences in spindle microtubule density. Scale bar, 5 μm, α-tubulin, and ch-TOG were stained with mouse monoclonal anti-α-tubulin and rabbit polyclonal anti-ch-TOG, respectively. ( B ) The bar graph shows the quantification of α-tubulin/MT intensity at the centrosomes (two). ( C ) The bar graph shows the percentage of cells (cells with above the average centrosomal microtubule intensity of TACC3 WT-expressed cells) with increased microtubule density phenotype in both the mutant cases. ( D ) The bar graph shows the quantification of ch-TOG intensity at the centrosomes ( E ) The bar graph shows the quantification of GFP-TACC3 intensity at the centrosomes. ( F ) The bar graph shows the quantification of ch-TOG intensity on the mitotic spindles. The regions of interest (ROI) used for quantification are shown in panel (A). ( G ) The bar graph shows the percentage of cells (cells with above the average ch-TOG intensity of TACC3 WT-expressed cells) with decreased ch-TOG intensity at the centrosomes phenotype in both the mutant cases. ( H ) The bar graph shows the percentage of cells (cells with above the average ch-TOG intensity of TACC3 WT-expressed cells) with increased ch-TOG intensity on the spindles in both the mutant cases. The bars represent mean ± S.E. The number of mitotic cells counted = 90–100 each (four independent experiments). ****, P <0.0001, ***, P < 0.001, **, P <0.01, *, P <0.05.
    Figure Legend Snippet: ( A ) Representative confocal images of GFP-TACC3 (WT)-TACC3 shRNA (top panel), GFP-TACC3 (Δ678–681)-TACC3 shRNA (middle panel), or GFP-TACC3 (Δ682–688)-TACC3 shRNA (bottom panel) transfected mitotic HeLa Kyoto cells showing the differences in spindle microtubule density. Scale bar, 5 μm, α-tubulin, and ch-TOG were stained with mouse monoclonal anti-α-tubulin and rabbit polyclonal anti-ch-TOG, respectively. ( B ) The bar graph shows the quantification of α-tubulin/MT intensity at the centrosomes (two). ( C ) The bar graph shows the percentage of cells (cells with above the average centrosomal microtubule intensity of TACC3 WT-expressed cells) with increased microtubule density phenotype in both the mutant cases. ( D ) The bar graph shows the quantification of ch-TOG intensity at the centrosomes ( E ) The bar graph shows the quantification of GFP-TACC3 intensity at the centrosomes. ( F ) The bar graph shows the quantification of ch-TOG intensity on the mitotic spindles. The regions of interest (ROI) used for quantification are shown in panel (A). ( G ) The bar graph shows the percentage of cells (cells with above the average ch-TOG intensity of TACC3 WT-expressed cells) with decreased ch-TOG intensity at the centrosomes phenotype in both the mutant cases. ( H ) The bar graph shows the percentage of cells (cells with above the average ch-TOG intensity of TACC3 WT-expressed cells) with increased ch-TOG intensity on the spindles in both the mutant cases. The bars represent mean ± S.E. The number of mitotic cells counted = 90–100 each (four independent experiments). ****, P <0.0001, ***, P < 0.001, **, P <0.01, *, P <0.05.

    Techniques Used: shRNA, Transfection, Staining, Mutagenesis

    ( A ) Immunoprecipitation of GFP-tagged TACC3 proteins using GFP antibody in lysates of HeLa Kyoto cells transfected with GFP-TACC3 (WT)-TACC3 shRNA, GFP-TACC3 (Δ678–681)-TACC3 shRNA, or GFP-TACC3 (Δ682–688)-TACC3 shRNA. The immunoblots were probed with GFP-TACC3 (WT), GFP-TACC3 (Δ678–681), and GFP-TACC3 (Δ682–688) forms along with γ-tubulin, GCP3, GCP4, and GCP6 by using the respective antibodies. ( B ) Fold change of different γ-TuRC proteins from the GFP-TACC3 immunoprecipitates of GFP-TACC3 WT, GFP-TACC3 (Δ678–681), or GFP-TACC3 (Δ682–688) expressed cells are plotted (based on three experiments in each). Data are mean ± S.E. ****, P <0.0001, ***, P <0.001, **, P <0.01, ns: not significant.
    Figure Legend Snippet: ( A ) Immunoprecipitation of GFP-tagged TACC3 proteins using GFP antibody in lysates of HeLa Kyoto cells transfected with GFP-TACC3 (WT)-TACC3 shRNA, GFP-TACC3 (Δ678–681)-TACC3 shRNA, or GFP-TACC3 (Δ682–688)-TACC3 shRNA. The immunoblots were probed with GFP-TACC3 (WT), GFP-TACC3 (Δ678–681), and GFP-TACC3 (Δ682–688) forms along with γ-tubulin, GCP3, GCP4, and GCP6 by using the respective antibodies. ( B ) Fold change of different γ-TuRC proteins from the GFP-TACC3 immunoprecipitates of GFP-TACC3 WT, GFP-TACC3 (Δ678–681), or GFP-TACC3 (Δ682–688) expressed cells are plotted (based on three experiments in each). Data are mean ± S.E. ****, P <0.0001, ***, P <0.001, **, P <0.01, ns: not significant.

    Techniques Used: Immunoprecipitation, Transfection, shRNA, Western Blot

    ( A ) Schematic representation of GFP-TACC3 (WT) and GFP-TACC3 ΔC (1–590) constructs. Position of Ser 558 phosphorylation site is also shown. ( B ) Lysates of HeLa Kyoto cells transfected with GFP-TACC3 (WT) along with TACC3 3′-UTR siRNA and GFP-TACC3 ΔC (1–590) along with TACC3 esiRNA for >48 h were analyzed by Western blot to detect the levels of exogenous TACC3 proteins and the depletion of endogenous TACC3 by the respective siRNAs. Both endogenous TACC3 and the exogenous TACC3 proteins were probed with mouse monoclonal anti-TACC3 antibody. α-tubulin was probed as a control. ( C ) Representative confocal images of GFP-TACC3 (WT) and GFP-TACC3 ΔC (1–590) transfected mitotic HeLa Kyoto cells showing the differences in γ-tubulin intensity. Scale bar, 5 μm, ( D ) The plots show γ-tubulin intensity at the centrosomes in different conditions as indicated. Centrosomal ROI used for quantification for both cases is shown. ( E ) The bar graph shows the percentage of cells (cells with above the average centrosomal γ-tubulin intensity of TACC3 WT-expressed cells) with decreased γ-tubulin intensity phenotype in GFP-TACC3 ΔC cells. The bars represent mean ± S.E. The number of mitotic cells counted = 60 each (four independent experiments). ****, P <0.0001, ***, P <0.001. ( F ) Immunoprecipitation of GFP-tagged TACC3 ΔC using the anti-GFP antibody in the lysate of HEK293T cells transfected with GFP-TACC3 ΔC. The immunoblots were probed with GFP-TACC3 ΔC, γ-tubulin, GCP3, GCP4, and GCP6 using the respective antibodies. ( G ) Immunoprecipitation of γ-tubulin using anti-γ-tubulin antibody in the lysate of HEK cells transfected with GFP-TACC3 ΔC. The immunoblots were probed with GFP-TACC3 ΔC and γ-tubulin using the respective antibodies. ( H ) Immunoprecipitation of GCP4 using anti-GCP4 antibody in the lysate of HEK293T cells transfected with GFP-TACC3 ΔC. The immunoblots were probed for GFP-TACC3 ΔC along with GCP4 by using respective antibodies.
    Figure Legend Snippet: ( A ) Schematic representation of GFP-TACC3 (WT) and GFP-TACC3 ΔC (1–590) constructs. Position of Ser 558 phosphorylation site is also shown. ( B ) Lysates of HeLa Kyoto cells transfected with GFP-TACC3 (WT) along with TACC3 3′-UTR siRNA and GFP-TACC3 ΔC (1–590) along with TACC3 esiRNA for >48 h were analyzed by Western blot to detect the levels of exogenous TACC3 proteins and the depletion of endogenous TACC3 by the respective siRNAs. Both endogenous TACC3 and the exogenous TACC3 proteins were probed with mouse monoclonal anti-TACC3 antibody. α-tubulin was probed as a control. ( C ) Representative confocal images of GFP-TACC3 (WT) and GFP-TACC3 ΔC (1–590) transfected mitotic HeLa Kyoto cells showing the differences in γ-tubulin intensity. Scale bar, 5 μm, ( D ) The plots show γ-tubulin intensity at the centrosomes in different conditions as indicated. Centrosomal ROI used for quantification for both cases is shown. ( E ) The bar graph shows the percentage of cells (cells with above the average centrosomal γ-tubulin intensity of TACC3 WT-expressed cells) with decreased γ-tubulin intensity phenotype in GFP-TACC3 ΔC cells. The bars represent mean ± S.E. The number of mitotic cells counted = 60 each (four independent experiments). ****, P <0.0001, ***, P <0.001. ( F ) Immunoprecipitation of GFP-tagged TACC3 ΔC using the anti-GFP antibody in the lysate of HEK293T cells transfected with GFP-TACC3 ΔC. The immunoblots were probed with GFP-TACC3 ΔC, γ-tubulin, GCP3, GCP4, and GCP6 using the respective antibodies. ( G ) Immunoprecipitation of γ-tubulin using anti-γ-tubulin antibody in the lysate of HEK cells transfected with GFP-TACC3 ΔC. The immunoblots were probed with GFP-TACC3 ΔC and γ-tubulin using the respective antibodies. ( H ) Immunoprecipitation of GCP4 using anti-GCP4 antibody in the lysate of HEK293T cells transfected with GFP-TACC3 ΔC. The immunoblots were probed for GFP-TACC3 ΔC along with GCP4 by using respective antibodies.

    Techniques Used: Construct, Transfection, esiRNA, Western Blot, Immunoprecipitation

    ( A ) Lysates of HeLa Kyoto cells transfected with GFP-TACC3 (WT)-TACC3 shRNA, GFP-TACC3 (Δ682–688)-TACC3 shRNA, or GFP-TACC3 (Δ678–681)-TACC3 shRNA for 48 h were analyzed by Western blot to detect the levels of GFP-TACC3 proteins with simultaneously probing for endogenous TACC3. Both endogenous TACC3 and GFP-TACC3 proteins were probed with mouse monoclonal anti-TACC3 antibody. Phospho-Ser 558 TACC3 was detected using rabbit monoclonal anti-phospho-Ser 558 TACC3 antibody. α-tubulin was probed as a control. ( B ) Representative confocal images of GFP-TACC3 (WT)-TACC3 shRNA (top panel), GFP-TACC3 (Δ678–681)-TACC3 shRNA (middle panel), or GFP-TACC3 (Δ682–688)-TACC3 shRNA (bottom panel)-transfected mitotic HeLa Kyoto cells showing the localization of phospho-Ser 558 TACC3. Scale bar, 5 μm. Phospho-Ser 558 TACC3 was detected using rabbit monoclonal anti-phospho-Ser 558 TACC3 antibody. ( C ) The bar graph shows intensity of phospho-Ser 558 TACC3 at centrosomes in synchronized metaphase cells in ROI (19.47 μm 2 ), as shown, around the centrosomes in different conditions as in panel (B). ( D ) The bar graph shows the percentage of cells (cells with above the average centrosomal phospho-Ser 558 TACC3 intensity of TACC3 WT-expressed cells) showing increased phospho-Ser 558 TACC3 intensity at the centrosomes in both the mutant cases. ( E ) The bar graph shows the quantification of phospho-Ser 558 TACC3 intensity on the mitotic spindles.The ROI used for quantification is shown in (b). ( F ) The bar graph shows the percentage of cells (cells with above the average phospho-Ser 558 TACC3 intensity of TACC3 WT-expressed cells) showing decreased phospho-Ser 558 TACC3 intensity on the spindles in both the mutant cases. The bars represent mean ± S.E. The number of cells counted = 70 each (four independent experiments) ****, P <0.0001, ***, P <0.001.
    Figure Legend Snippet: ( A ) Lysates of HeLa Kyoto cells transfected with GFP-TACC3 (WT)-TACC3 shRNA, GFP-TACC3 (Δ682–688)-TACC3 shRNA, or GFP-TACC3 (Δ678–681)-TACC3 shRNA for 48 h were analyzed by Western blot to detect the levels of GFP-TACC3 proteins with simultaneously probing for endogenous TACC3. Both endogenous TACC3 and GFP-TACC3 proteins were probed with mouse monoclonal anti-TACC3 antibody. Phospho-Ser 558 TACC3 was detected using rabbit monoclonal anti-phospho-Ser 558 TACC3 antibody. α-tubulin was probed as a control. ( B ) Representative confocal images of GFP-TACC3 (WT)-TACC3 shRNA (top panel), GFP-TACC3 (Δ678–681)-TACC3 shRNA (middle panel), or GFP-TACC3 (Δ682–688)-TACC3 shRNA (bottom panel)-transfected mitotic HeLa Kyoto cells showing the localization of phospho-Ser 558 TACC3. Scale bar, 5 μm. Phospho-Ser 558 TACC3 was detected using rabbit monoclonal anti-phospho-Ser 558 TACC3 antibody. ( C ) The bar graph shows intensity of phospho-Ser 558 TACC3 at centrosomes in synchronized metaphase cells in ROI (19.47 μm 2 ), as shown, around the centrosomes in different conditions as in panel (B). ( D ) The bar graph shows the percentage of cells (cells with above the average centrosomal phospho-Ser 558 TACC3 intensity of TACC3 WT-expressed cells) showing increased phospho-Ser 558 TACC3 intensity at the centrosomes in both the mutant cases. ( E ) The bar graph shows the quantification of phospho-Ser 558 TACC3 intensity on the mitotic spindles.The ROI used for quantification is shown in (b). ( F ) The bar graph shows the percentage of cells (cells with above the average phospho-Ser 558 TACC3 intensity of TACC3 WT-expressed cells) showing decreased phospho-Ser 558 TACC3 intensity on the spindles in both the mutant cases. The bars represent mean ± S.E. The number of cells counted = 70 each (four independent experiments) ****, P <0.0001, ***, P <0.001.

    Techniques Used: Transfection, shRNA, Western Blot, Mutagenesis

    ( A ) Lysates of HeLa Kyoto cells transfected with control siRNA and ch-TOG siRNA for 48 h were analyzed by Western blot to detect ch-TOG expression. ( B ) Representative confocal images of control siRNA or ch-TOG siRNA transfected mitotic Hela Kyoto cells showing the γ-tubulin intensity at the centrosomes and mitotic spindle. Scale bar, 5 μm. γ-tubulin was stained with mouse monoclonal anti-γ-tubulin antibody. Centrosomal regions are shown in an enlarged view. ( C ) The bar graph shows the quantification of centrosomal γ-tubulin intensity as of (b). The bars represent mean ± S.E. The number of cells counted = 50 each (four independent experiments). ( D ) The bar graph shows the quantification of γ-tubulin intensity on the spindles in different conditions as of (b). ( E ) The bar graph shows the percentage of cells (cells with above the average γ-tubulin intensity of TACC3 WT-expressed cells) showing increased γ-tubulin intensity at the spindles phenotype in ch-TOG siRNA treated cells. The bars represent mean ± S.E. The number of cells counted = 50 each (four independent experiments) ****, P <0.0001, ***, P <0.001. ( F ) Immunoprecipitation of GFP tagged ch-TOG using GFP coated beads (GFP-Trap) in the lysates of HeLa Kyoto cells stably expressing GFP-ch-TOG or control HeLa cells transfected with an empty GFP-vector (pcDNA3-EGFP). The immunoblots were probed for GFP-ch-TOG, γ-tubulin, GCP3, GCP4, GCP6, and TACC3 using the respective antibodies. ( G ) Immunoprecipitation of GFP-tagged ch-TOG C-terminus (1428–2032) using anti-GFP antibody in the lysates of HEK cells transfected with GFP-ch-TOG C-ter. The immunoblots were probed for GFP-ch-TOG, γ-tubulin, GCP3, GCP4, and TACC3 using the respective antibodies.
    Figure Legend Snippet: ( A ) Lysates of HeLa Kyoto cells transfected with control siRNA and ch-TOG siRNA for 48 h were analyzed by Western blot to detect ch-TOG expression. ( B ) Representative confocal images of control siRNA or ch-TOG siRNA transfected mitotic Hela Kyoto cells showing the γ-tubulin intensity at the centrosomes and mitotic spindle. Scale bar, 5 μm. γ-tubulin was stained with mouse monoclonal anti-γ-tubulin antibody. Centrosomal regions are shown in an enlarged view. ( C ) The bar graph shows the quantification of centrosomal γ-tubulin intensity as of (b). The bars represent mean ± S.E. The number of cells counted = 50 each (four independent experiments). ( D ) The bar graph shows the quantification of γ-tubulin intensity on the spindles in different conditions as of (b). ( E ) The bar graph shows the percentage of cells (cells with above the average γ-tubulin intensity of TACC3 WT-expressed cells) showing increased γ-tubulin intensity at the spindles phenotype in ch-TOG siRNA treated cells. The bars represent mean ± S.E. The number of cells counted = 50 each (four independent experiments) ****, P <0.0001, ***, P <0.001. ( F ) Immunoprecipitation of GFP tagged ch-TOG using GFP coated beads (GFP-Trap) in the lysates of HeLa Kyoto cells stably expressing GFP-ch-TOG or control HeLa cells transfected with an empty GFP-vector (pcDNA3-EGFP). The immunoblots were probed for GFP-ch-TOG, γ-tubulin, GCP3, GCP4, GCP6, and TACC3 using the respective antibodies. ( G ) Immunoprecipitation of GFP-tagged ch-TOG C-terminus (1428–2032) using anti-GFP antibody in the lysates of HEK cells transfected with GFP-ch-TOG C-ter. The immunoblots were probed for GFP-ch-TOG, γ-tubulin, GCP3, GCP4, and TACC3 using the respective antibodies.

    Techniques Used: Transfection, Western Blot, Expressing, Staining, Immunoprecipitation, Stable Transfection, Plasmid Preparation

    rabbit anti actb polyclonal igg  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit anti actb polyclonal igg
    Rabbit Anti Actb Polyclonal Igg, 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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    rabbit polyclonal anti atg16l1  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit polyclonal anti atg16l1
    (A) Western blotting and Coomassie staining of GFP-IPs from MCF10A GFP-LC3A expressing cells treated +/- LLOMe (250 µM, 40 mins). (B) Normalized mass spectrometry analysis of LC3A-PE and LC3A-PS in cells treated as in (A). (C) Representative confocal images of GFP-LC3A in wild type and ATG13 KO MCF10A cells treated with LLOMe (250 µM, 30 min) or PP242 (1 µM, 1 hr), after pre-treatment with BafA1 (100 nM). Scale bar, 10 µm. (D) Representative confocal images of endogenous LC3 in wild type MCF10A cells treated as in (C). Scale bar, 10 µm. (E) Confocal images of wild type or ATG13 KO MCF10A cells expressing GFP-LC3A treated with LLOMe (250 µM, 20 min) +/- BafA1 pre-treatment (100 nM) and stained for Galectin-3. Inserts show single channel cropped images. Scale bar, 5 µm. MCF10A cells were treated with monensin (100 μM), SaliP (2.5 μM), PP242 (1 µM), LLOMe (250 μM) or LLOMe + BafA1 (100 nM) for 1 h. Following fractionation, membrane and cytosol fractions were probed for ATP6V1A, ATP6V0D1 by western blotting. V1/V0 ratios shown below. (G) Western blotting analysis of GFP-LC3A expressing wild type MCF10A and <t>ATG16L1</t> KO cells re-expressing ATG16L1 WT or K490A, treated with LLOMe (250 µM, 20 min). (H) Confocal images of GFP-LC3A in the MCF10A ATG16L1 cell line panel treated with LLOMe (250 µM, 20 mins) or MSU crystals (200 µg/ml, 4 hr) and co stained for LAMP1. Arrows denote LAMP1 positive crystals. Scale bar, 10 µm. (I) Confocal images of GFP-LC3A expressing MCF10A cells co-transfected with mCherry-SopF. Outlined cells mark mCherry-SopF expressing cells. Scale bar, 20 µm.
    Rabbit Polyclonal Anti Atg16l1, 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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    Images

    1) Product Images from "Lysosome damage triggers direct ATG8 conjugation and ATG2 engagement via CASM"

    Article Title: Lysosome damage triggers direct ATG8 conjugation and ATG2 engagement via CASM

    Journal: bioRxiv

    doi: 10.1101/2023.03.22.533754

    (A) Western blotting and Coomassie staining of GFP-IPs from MCF10A GFP-LC3A expressing cells treated +/- LLOMe (250 µM, 40 mins). (B) Normalized mass spectrometry analysis of LC3A-PE and LC3A-PS in cells treated as in (A). (C) Representative confocal images of GFP-LC3A in wild type and ATG13 KO MCF10A cells treated with LLOMe (250 µM, 30 min) or PP242 (1 µM, 1 hr), after pre-treatment with BafA1 (100 nM). Scale bar, 10 µm. (D) Representative confocal images of endogenous LC3 in wild type MCF10A cells treated as in (C). Scale bar, 10 µm. (E) Confocal images of wild type or ATG13 KO MCF10A cells expressing GFP-LC3A treated with LLOMe (250 µM, 20 min) +/- BafA1 pre-treatment (100 nM) and stained for Galectin-3. Inserts show single channel cropped images. Scale bar, 5 µm. MCF10A cells were treated with monensin (100 μM), SaliP (2.5 μM), PP242 (1 µM), LLOMe (250 μM) or LLOMe + BafA1 (100 nM) for 1 h. Following fractionation, membrane and cytosol fractions were probed for ATP6V1A, ATP6V0D1 by western blotting. V1/V0 ratios shown below. (G) Western blotting analysis of GFP-LC3A expressing wild type MCF10A and ATG16L1 KO cells re-expressing ATG16L1 WT or K490A, treated with LLOMe (250 µM, 20 min). (H) Confocal images of GFP-LC3A in the MCF10A ATG16L1 cell line panel treated with LLOMe (250 µM, 20 mins) or MSU crystals (200 µg/ml, 4 hr) and co stained for LAMP1. Arrows denote LAMP1 positive crystals. Scale bar, 10 µm. (I) Confocal images of GFP-LC3A expressing MCF10A cells co-transfected with mCherry-SopF. Outlined cells mark mCherry-SopF expressing cells. Scale bar, 20 µm.
    Figure Legend Snippet: (A) Western blotting and Coomassie staining of GFP-IPs from MCF10A GFP-LC3A expressing cells treated +/- LLOMe (250 µM, 40 mins). (B) Normalized mass spectrometry analysis of LC3A-PE and LC3A-PS in cells treated as in (A). (C) Representative confocal images of GFP-LC3A in wild type and ATG13 KO MCF10A cells treated with LLOMe (250 µM, 30 min) or PP242 (1 µM, 1 hr), after pre-treatment with BafA1 (100 nM). Scale bar, 10 µm. (D) Representative confocal images of endogenous LC3 in wild type MCF10A cells treated as in (C). Scale bar, 10 µm. (E) Confocal images of wild type or ATG13 KO MCF10A cells expressing GFP-LC3A treated with LLOMe (250 µM, 20 min) +/- BafA1 pre-treatment (100 nM) and stained for Galectin-3. Inserts show single channel cropped images. Scale bar, 5 µm. MCF10A cells were treated with monensin (100 μM), SaliP (2.5 μM), PP242 (1 µM), LLOMe (250 μM) or LLOMe + BafA1 (100 nM) for 1 h. Following fractionation, membrane and cytosol fractions were probed for ATP6V1A, ATP6V0D1 by western blotting. V1/V0 ratios shown below. (G) Western blotting analysis of GFP-LC3A expressing wild type MCF10A and ATG16L1 KO cells re-expressing ATG16L1 WT or K490A, treated with LLOMe (250 µM, 20 min). (H) Confocal images of GFP-LC3A in the MCF10A ATG16L1 cell line panel treated with LLOMe (250 µM, 20 mins) or MSU crystals (200 µg/ml, 4 hr) and co stained for LAMP1. Arrows denote LAMP1 positive crystals. Scale bar, 10 µm. (I) Confocal images of GFP-LC3A expressing MCF10A cells co-transfected with mCherry-SopF. Outlined cells mark mCherry-SopF expressing cells. Scale bar, 20 µm.

    Techniques Used: Western Blot, Staining, Expressing, Mass Spectrometry, Fractionation, Transfection

    (A) Western blot analysis of GFP-IPs and input from wild type and ATG13 KO MCF10A cells expressing either GFP or GFP-LC3A treated +/- LLOMe (250 µM, 30 mins). Samples probed for ATG2B, GFP, ATG13 and GAPDH. (B) Western blot analysis of GFP-IPs and input from MCF10A ATG16L1 cell line panel expressing GFP-LC3A treated +/- LLOMe (250 µM, 30 mins). Samples probed for ATG2B, GFP, ATG16L1 and GAPDH. (C) Western blot analysis of GFP-IPs and total cell lysate (TCL) from GFP-LC3A expressing MCF10A cells treated with LLOMe (250 µM, 30 mins) or PP242 (1 µM, 1 hr) +/- Vps34 IN-1 (5 µM). Samples probed for ATG2B, GFP and GAPDH. (D) GFP-LC3A expressing ATG13 or ATG16L1 KO MCF10A cells were treated with LLOMe (250 µM, 30 mins). Following fractionation, input, cytosol and membrane fractions were probed for ATG2B, LAMP1, GFP and β-tubulin. (E) Western blot analysis of GFP-IPs and input from ATG13 KO MCF10A cells expressing GFP-LC3A treated with LLOMe (250 µM, 30mins), GPN (200 µM, 30 min) or monensin (100 µM, 40 mins). Samples probed for ATG2B and GFP. (F) Western blot analysis of GFP-IPs and input from RAW264.7 cells expressing GFP-LC3A following incubation with opsonized zymosan (OPZ) for 40 mins. Samples were probed for ATG2B and GFP. (G) Model of ATG8 response to lysosome damage illustrating the ATG8 associated lysosome tubulation and vesiculation, activation of the V-ATPase-ATG16L1 axis, ATG8 conjugation to PE and PS, and the engagement of lipid transfer protein ATG2. Image created using Biorender.
    Figure Legend Snippet: (A) Western blot analysis of GFP-IPs and input from wild type and ATG13 KO MCF10A cells expressing either GFP or GFP-LC3A treated +/- LLOMe (250 µM, 30 mins). Samples probed for ATG2B, GFP, ATG13 and GAPDH. (B) Western blot analysis of GFP-IPs and input from MCF10A ATG16L1 cell line panel expressing GFP-LC3A treated +/- LLOMe (250 µM, 30 mins). Samples probed for ATG2B, GFP, ATG16L1 and GAPDH. (C) Western blot analysis of GFP-IPs and total cell lysate (TCL) from GFP-LC3A expressing MCF10A cells treated with LLOMe (250 µM, 30 mins) or PP242 (1 µM, 1 hr) +/- Vps34 IN-1 (5 µM). Samples probed for ATG2B, GFP and GAPDH. (D) GFP-LC3A expressing ATG13 or ATG16L1 KO MCF10A cells were treated with LLOMe (250 µM, 30 mins). Following fractionation, input, cytosol and membrane fractions were probed for ATG2B, LAMP1, GFP and β-tubulin. (E) Western blot analysis of GFP-IPs and input from ATG13 KO MCF10A cells expressing GFP-LC3A treated with LLOMe (250 µM, 30mins), GPN (200 µM, 30 min) or monensin (100 µM, 40 mins). Samples probed for ATG2B and GFP. (F) Western blot analysis of GFP-IPs and input from RAW264.7 cells expressing GFP-LC3A following incubation with opsonized zymosan (OPZ) for 40 mins. Samples were probed for ATG2B and GFP. (G) Model of ATG8 response to lysosome damage illustrating the ATG8 associated lysosome tubulation and vesiculation, activation of the V-ATPase-ATG16L1 axis, ATG8 conjugation to PE and PS, and the engagement of lipid transfer protein ATG2. Image created using Biorender.

    Techniques Used: Western Blot, Expressing, Fractionation, Incubation, Activation Assay, Conjugation Assay

    rabbit anti actb polyclonal igg  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit anti actb polyclonal igg
    Rabbit Anti Actb Polyclonal Igg, 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 actb polyclonal igg/product/Cell Signaling Technology Inc
    Average 86 stars, based on 1 article reviews
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    chicken polyclonal anti ul34  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc chicken polyclonal anti ul34
    Nuclear egress phenotypes comparison of pUL13 K176A and pUS3 mutants (A-H) Shown are digital confocal images representing the localization of <t>UL34</t> in infected Vero cells Green: pUL34. Cells were infected with HSV-1(F) (BAC), US3 K220A , UL13 K176A , and US3 mutants for 16 h at a MOI of 5, fixed at 16 hpi, and stained using antibodies directed against pUL34. Scale bar = 10μm. Representative images of one of three independent experiments are shown. (I) Frequencies of infected cell nuclei that show NEC aggregates. The error bars show the range of the values. Statistical significance was determined by one-way analysis of variance (ANOVA) using the Tukey method for multiple comparisons. ns, not significant; ***, P < 0.001. Data on graph represents at least three independent experiments. (J) US3 mobility shift assay of UL13 K176A and US3 mutants. Digital images of immunoblots for the indicated proteins in lysates of Vero cells infected for 18 h with 5 PFU/cell of BAC-derived WT HSV-1(F), US3 K220A or pUL13 K176A or US3 mutants. UL34 serves as a marker of equivalent infection and actin as a loading control.
    Chicken Polyclonal Anti Ul34, 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 polyclonal anti ul34/product/Cell Signaling Technology Inc
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    Images

    1) Product Images from "DISTINCT ROLES OF VIRAL US3 AND UL13 PROTEIN KINASES IN HERPES VIRUS SIMPLEX TYPE 1 (HSV-1) NUCLEAR EGRESS"

    Article Title: DISTINCT ROLES OF VIRAL US3 AND UL13 PROTEIN KINASES IN HERPES VIRUS SIMPLEX TYPE 1 (HSV-1) NUCLEAR EGRESS

    Journal: bioRxiv

    doi: 10.1101/2023.03.20.533584

    Nuclear egress phenotypes comparison of pUL13 K176A and pUS3 mutants (A-H) Shown are digital confocal images representing the localization of UL34 in infected Vero cells Green: pUL34. Cells were infected with HSV-1(F) (BAC), US3 K220A , UL13 K176A , and US3 mutants for 16 h at a MOI of 5, fixed at 16 hpi, and stained using antibodies directed against pUL34. Scale bar = 10μm. Representative images of one of three independent experiments are shown. (I) Frequencies of infected cell nuclei that show NEC aggregates. The error bars show the range of the values. Statistical significance was determined by one-way analysis of variance (ANOVA) using the Tukey method for multiple comparisons. ns, not significant; ***, P < 0.001. Data on graph represents at least three independent experiments. (J) US3 mobility shift assay of UL13 K176A and US3 mutants. Digital images of immunoblots for the indicated proteins in lysates of Vero cells infected for 18 h with 5 PFU/cell of BAC-derived WT HSV-1(F), US3 K220A or pUL13 K176A or US3 mutants. UL34 serves as a marker of equivalent infection and actin as a loading control.
    Figure Legend Snippet: Nuclear egress phenotypes comparison of pUL13 K176A and pUS3 mutants (A-H) Shown are digital confocal images representing the localization of UL34 in infected Vero cells Green: pUL34. Cells were infected with HSV-1(F) (BAC), US3 K220A , UL13 K176A , and US3 mutants for 16 h at a MOI of 5, fixed at 16 hpi, and stained using antibodies directed against pUL34. Scale bar = 10μm. Representative images of one of three independent experiments are shown. (I) Frequencies of infected cell nuclei that show NEC aggregates. The error bars show the range of the values. Statistical significance was determined by one-way analysis of variance (ANOVA) using the Tukey method for multiple comparisons. ns, not significant; ***, P < 0.001. Data on graph represents at least three independent experiments. (J) US3 mobility shift assay of UL13 K176A and US3 mutants. Digital images of immunoblots for the indicated proteins in lysates of Vero cells infected for 18 h with 5 PFU/cell of BAC-derived WT HSV-1(F), US3 K220A or pUL13 K176A or US3 mutants. UL34 serves as a marker of equivalent infection and actin as a loading control.

    Techniques Used: Infection, Staining, Mobility Shift, Western Blot, Derivative Assay, Marker

    UL13 colocalizes with pUL31 inside the nucleus and interacts with pUL31 Shown are digital confocal images of representing the localization of FLAG-UL31, HA-UL34, and EGFP-UL13 transiently co-expressed proteins in Vero cells, single-cell transfection (A-C), without EGFP-UL13(D-G), or with EGFP-UL13 co-expression (H-K). Cells were fixed at 48 h post-transfection and were stained using antibodies directed against HA and FLAG. Green: EGFP-UL13, Blue: HA-UL34, Red: UL31-FLAG. Representative images of one of three independent experiments are shown. Bars, 5μm. (L) Co-immunoprecipitation of EGFP-UL13 and FLAG-UL31. 293-T cells were co-transfected with FLAG-UL31 and EGFP-UL13 plasmids. Cells were lysed after 48 hours post-transfection. Cell lysate samples were collected and incubated with FLAG magnetic beads to immunoprecipitate UL31 overnight. The whole lysate (WL) samples and immunoprecipitated (IP) eluents were separated by SDS-PAGE, blotted onto nitrocellulose membrane, and probed as indicated in the figure.
    Figure Legend Snippet: UL13 colocalizes with pUL31 inside the nucleus and interacts with pUL31 Shown are digital confocal images of representing the localization of FLAG-UL31, HA-UL34, and EGFP-UL13 transiently co-expressed proteins in Vero cells, single-cell transfection (A-C), without EGFP-UL13(D-G), or with EGFP-UL13 co-expression (H-K). Cells were fixed at 48 h post-transfection and were stained using antibodies directed against HA and FLAG. Green: EGFP-UL13, Blue: HA-UL34, Red: UL31-FLAG. Representative images of one of three independent experiments are shown. Bars, 5μm. (L) Co-immunoprecipitation of EGFP-UL13 and FLAG-UL31. 293-T cells were co-transfected with FLAG-UL31 and EGFP-UL13 plasmids. Cells were lysed after 48 hours post-transfection. Cell lysate samples were collected and incubated with FLAG magnetic beads to immunoprecipitate UL31 overnight. The whole lysate (WL) samples and immunoprecipitated (IP) eluents were separated by SDS-PAGE, blotted onto nitrocellulose membrane, and probed as indicated in the figure.

    Techniques Used: Transfection, Expressing, Staining, Immunoprecipitation, Incubation, Magnetic Beads, SDS Page

    rabbit polyclonal antibody anti rbp jκ  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit polyclonal antibody anti rbp jκ
    Rabbit Polyclonal Antibody Anti Rbp Jκ, 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 mouse anti β actin polyclonal antibody
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    Cell Signaling Technology Inc primary polyclonal antibodies targeting anti yap1
    (A and B) Representative images and analysis of immunohistochemistry staining for <t>YAP1</t> (×100) and YAP1(s127) (×200) in human HCC subgrouped as shown in the figure. ImageJ was used to analyze the images. (C–E) Linear regression analysis of the expression of RP11-40C6.2 and YAP1 (IOD value) or percentage of YAP1 nuclear translocation and YAP1 (s127) in human HCC. (F) Diagram of potential TEADs binding sites and mutation design within the RP11-40C6.2 promoter region. (G) Luciferase reporter assay of HBx, HBc, and LPA treatment on the activity of wild-type (WT) and TEADs binding site mutation (Mut) RP11-40C6.2 promoter. (H) Antibody to TEAD 1-4 was used in chromatin immunoprecipitation (ChIP) assays; p65 and IgG were negative controls. (I) Co-IP assays were performed to study the binding between TAZ and HBx or HBc; the protein complex was immuno-precipitated with TAZ antibody and further examined by HBx or HBc antibody. Mean±SEM values from at least three independent experiments are presented. * p <0.05, ** p <0.01.
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    Cell Signaling Technology Inc polyclonal rabbit abs
    Generation of chimeric human/mouse CD79 knockin mice. (A) Schematic representation of B cell Ag receptor complex. (B) CD79 amino acid conservation between mice and humans. Black intervals represent regions of 0% amino acid conservation. Domain demarcation: CY, cytoplasmic; EC, extracellular; L, leader; TM, transmembrane. Conserved cysteines required for interchain disulfide bonding shown for each. (C) Schematic representation of cCD79. Human sequences comprise the CD79B extracellular domain and CD79A extracellular/transmembrane domains. (D) Surface staining of splenic B cells (B220+) from chimeric (h/m)CD79 knockin mice and control C57BL/6 mice. Gray lines show B220−. (E) Rabbit anti-mCD79 immunoblot analysis of whole-cell lysates (purified splenic B cells, CD43−) from cCD79 and WT mice. Upper membrane probed with a <t>polyclonal</t> rabbit Ab raised against a mCD79A and mCD79B extracellular domain fusion protein (Cambier laboratory). Middle membrane probed with a polyclonal rabbit Ab raised against the cytoplasmic domain of mCD79B (Cambier laboratory). An anti–β actin blot was used as a protein loading control. (F) Surface staining of B cell gated (CD19+) human PBMCs with anti-hCD79B (AT-105). Gray line shows CD19−. (G) Surface staining as a function of cCD79B allele dosage. Splenocytes from cCD79 mice of the indicated genotypes were stained with both anti-hCD79B (AT-105) and anti-mCD79B (HM79). Gray contour shows B220−. (H) IgM and IgD surface expression in chimeric mice described in (G). Gray line shows B220−. n = 4 female mice per group for (G) and (H). Error bars represent SEM. All data represent at least three independent experiments; representative data are shown.
    Polyclonal Rabbit Abs, 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 rabbit polyclonal phospho tacc3 antibody
    ( A ) Schematic representation of <t>GFP-TACC3</t> (Δ678–681)-TACC3 shRNA and GFP-TACC3 (Δ682–688)-TACC3 shRNA constructs. ( B ) Lysates of HeLa Kyoto cells transfected with GFP-TACC3 (WT)-TACC3 shRNA, GFP-TACC3 (Δ682–688)-TACC3 shRNA, or GFP-TACC3 (Δ678–681)-TACC3 shRNA for 48 h were analyzed for exogenous TACC3 proteins with simultaneous depletion of endogenous TACC3. Both endogenous TACC3 and GFP-TACC3 proteins were stained with mouse monoclonal TACC3 antibody. Actin was probed as a control. TACC3 level in control untrasfected cells is also shown. ( C ) Immunoprecipitation of GFP-tagged TACC3 proteins using the anti-GFP antibody in the lysates of HeLa Kyoto cells transfected with GFP-TACC3 (WT)-TACC3 shRNA, GFP-TACC3 (Δ678–681)-TACC3 shRNA, or GFP-TACC3 (Δ682–688)-TACC3 shRNA. The immunoblots were probed with GFP and ch-TOG antibodies. ( D ) Representative confocal images of GFP-TACC3 (WT)-TACC3 shRNA (top panel), GFP-TACC3 (Δ678-681)-TACC3 shRNA (middle panel), or GFP-TACC3 (Δ682–688)-TACC3 shRNA (bottom panel)- transfected mitotic HeLa Kyoto cells showing the differences in γ-tubulin levels at the centrosomes. Scale bar, 5 μm, γ-tubulin, and ch-TOG were stained with mouse monoclonal anti-γ-tubulin and rabbit polyclonal anti-ch-TOG antibody, respectively. ( E ) The bar graph shows the quantification of γ-tubulin intensity at the centrosomes (two) in different conditions as of (d). ( F ) Representative confocal images of GFP-TACC3 (WT)-TACC3 shRNA (top panel), GFP-TACC3 (Δ678–681)-TACC3 shRNA (middle panel), or GFP-TACC3 (Δ682–688)-TACC3 shRNA (bottom panel) transfected mitotic HeLa Kyoto cells showing the differences in GCP6 localization at centrosomes. Scale bar, 5 μm, GCP6 was stained with mouse monoclonal anti-GCP6 antibody. ( G ) The bar graph shows the quantification of GCP6 intensity at the centrosomes in different conditions as in panel (F). ( H ) The bar graph shows the percentage of cells (cells with above the average centrosomal γ-tubulin intensity of TACC3 WT-expressed cells) with increased γ-tubulin intensity phenotype in both the mutant cases. The bars in (e), (g), and (h) represent mean ± S.E. The number of mitotic cells counted = 100 each (four independent experiments); ****, P <0.0001, ***, P <0.001.
    Rabbit Polyclonal Phospho Tacc3 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
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    Cell Signaling Technology Inc rabbit anti actb polyclonal igg
    ( A ) Schematic representation of <t>GFP-TACC3</t> (Δ678–681)-TACC3 shRNA and GFP-TACC3 (Δ682–688)-TACC3 shRNA constructs. ( B ) Lysates of HeLa Kyoto cells transfected with GFP-TACC3 (WT)-TACC3 shRNA, GFP-TACC3 (Δ682–688)-TACC3 shRNA, or GFP-TACC3 (Δ678–681)-TACC3 shRNA for 48 h were analyzed for exogenous TACC3 proteins with simultaneous depletion of endogenous TACC3. Both endogenous TACC3 and GFP-TACC3 proteins were stained with mouse monoclonal TACC3 antibody. Actin was probed as a control. TACC3 level in control untrasfected cells is also shown. ( C ) Immunoprecipitation of GFP-tagged TACC3 proteins using the anti-GFP antibody in the lysates of HeLa Kyoto cells transfected with GFP-TACC3 (WT)-TACC3 shRNA, GFP-TACC3 (Δ678–681)-TACC3 shRNA, or GFP-TACC3 (Δ682–688)-TACC3 shRNA. The immunoblots were probed with GFP and ch-TOG antibodies. ( D ) Representative confocal images of GFP-TACC3 (WT)-TACC3 shRNA (top panel), GFP-TACC3 (Δ678-681)-TACC3 shRNA (middle panel), or GFP-TACC3 (Δ682–688)-TACC3 shRNA (bottom panel)- transfected mitotic HeLa Kyoto cells showing the differences in γ-tubulin levels at the centrosomes. Scale bar, 5 μm, γ-tubulin, and ch-TOG were stained with mouse monoclonal anti-γ-tubulin and rabbit polyclonal anti-ch-TOG antibody, respectively. ( E ) The bar graph shows the quantification of γ-tubulin intensity at the centrosomes (two) in different conditions as of (d). ( F ) Representative confocal images of GFP-TACC3 (WT)-TACC3 shRNA (top panel), GFP-TACC3 (Δ678–681)-TACC3 shRNA (middle panel), or GFP-TACC3 (Δ682–688)-TACC3 shRNA (bottom panel) transfected mitotic HeLa Kyoto cells showing the differences in GCP6 localization at centrosomes. Scale bar, 5 μm, GCP6 was stained with mouse monoclonal anti-GCP6 antibody. ( G ) The bar graph shows the quantification of GCP6 intensity at the centrosomes in different conditions as in panel (F). ( H ) The bar graph shows the percentage of cells (cells with above the average centrosomal γ-tubulin intensity of TACC3 WT-expressed cells) with increased γ-tubulin intensity phenotype in both the mutant cases. The bars in (e), (g), and (h) represent mean ± S.E. The number of mitotic cells counted = 100 each (four independent experiments); ****, P <0.0001, ***, P <0.001.
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    Cell Signaling Technology Inc rabbit polyclonal anti atg16l1
    (A) Western blotting and Coomassie staining of GFP-IPs from MCF10A GFP-LC3A expressing cells treated +/- LLOMe (250 µM, 40 mins). (B) Normalized mass spectrometry analysis of LC3A-PE and LC3A-PS in cells treated as in (A). (C) Representative confocal images of GFP-LC3A in wild type and ATG13 KO MCF10A cells treated with LLOMe (250 µM, 30 min) or PP242 (1 µM, 1 hr), after pre-treatment with BafA1 (100 nM). Scale bar, 10 µm. (D) Representative confocal images of endogenous LC3 in wild type MCF10A cells treated as in (C). Scale bar, 10 µm. (E) Confocal images of wild type or ATG13 KO MCF10A cells expressing GFP-LC3A treated with LLOMe (250 µM, 20 min) +/- BafA1 pre-treatment (100 nM) and stained for Galectin-3. Inserts show single channel cropped images. Scale bar, 5 µm. MCF10A cells were treated with monensin (100 μM), SaliP (2.5 μM), PP242 (1 µM), LLOMe (250 μM) or LLOMe + BafA1 (100 nM) for 1 h. Following fractionation, membrane and cytosol fractions were probed for ATP6V1A, ATP6V0D1 by western blotting. V1/V0 ratios shown below. (G) Western blotting analysis of GFP-LC3A expressing wild type MCF10A and <t>ATG16L1</t> KO cells re-expressing ATG16L1 WT or K490A, treated with LLOMe (250 µM, 20 min). (H) Confocal images of GFP-LC3A in the MCF10A ATG16L1 cell line panel treated with LLOMe (250 µM, 20 mins) or MSU crystals (200 µg/ml, 4 hr) and co stained for LAMP1. Arrows denote LAMP1 positive crystals. Scale bar, 10 µm. (I) Confocal images of GFP-LC3A expressing MCF10A cells co-transfected with mCherry-SopF. Outlined cells mark mCherry-SopF expressing cells. Scale bar, 20 µm.
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    Cell Signaling Technology Inc chicken polyclonal anti ul34
    Nuclear egress phenotypes comparison of pUL13 K176A and pUS3 mutants (A-H) Shown are digital confocal images representing the localization of <t>UL34</t> in infected Vero cells Green: pUL34. Cells were infected with HSV-1(F) (BAC), US3 K220A , UL13 K176A , and US3 mutants for 16 h at a MOI of 5, fixed at 16 hpi, and stained using antibodies directed against pUL34. Scale bar = 10μm. Representative images of one of three independent experiments are shown. (I) Frequencies of infected cell nuclei that show NEC aggregates. The error bars show the range of the values. Statistical significance was determined by one-way analysis of variance (ANOVA) using the Tukey method for multiple comparisons. ns, not significant; ***, P < 0.001. Data on graph represents at least three independent experiments. (J) US3 mobility shift assay of UL13 K176A and US3 mutants. Digital images of immunoblots for the indicated proteins in lysates of Vero cells infected for 18 h with 5 PFU/cell of BAC-derived WT HSV-1(F), US3 K220A or pUL13 K176A or US3 mutants. UL34 serves as a marker of equivalent infection and actin as a loading control.
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    Cell Signaling Technology Inc rabbit polyclonal antibody anti rbp jκ
    Nuclear egress phenotypes comparison of pUL13 K176A and pUS3 mutants (A-H) Shown are digital confocal images representing the localization of <t>UL34</t> in infected Vero cells Green: pUL34. Cells were infected with HSV-1(F) (BAC), US3 K220A , UL13 K176A , and US3 mutants for 16 h at a MOI of 5, fixed at 16 hpi, and stained using antibodies directed against pUL34. Scale bar = 10μm. Representative images of one of three independent experiments are shown. (I) Frequencies of infected cell nuclei that show NEC aggregates. The error bars show the range of the values. Statistical significance was determined by one-way analysis of variance (ANOVA) using the Tukey method for multiple comparisons. ns, not significant; ***, P < 0.001. Data on graph represents at least three independent experiments. (J) US3 mobility shift assay of UL13 K176A and US3 mutants. Digital images of immunoblots for the indicated proteins in lysates of Vero cells infected for 18 h with 5 PFU/cell of BAC-derived WT HSV-1(F), US3 K220A or pUL13 K176A or US3 mutants. UL34 serves as a marker of equivalent infection and actin as a loading control.
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    Image Search Results


    (A and B) Representative images and analysis of immunohistochemistry staining for YAP1 (×100) and YAP1(s127) (×200) in human HCC subgrouped as shown in the figure. ImageJ was used to analyze the images. (C–E) Linear regression analysis of the expression of RP11-40C6.2 and YAP1 (IOD value) or percentage of YAP1 nuclear translocation and YAP1 (s127) in human HCC. (F) Diagram of potential TEADs binding sites and mutation design within the RP11-40C6.2 promoter region. (G) Luciferase reporter assay of HBx, HBc, and LPA treatment on the activity of wild-type (WT) and TEADs binding site mutation (Mut) RP11-40C6.2 promoter. (H) Antibody to TEAD 1-4 was used in chromatin immunoprecipitation (ChIP) assays; p65 and IgG were negative controls. (I) Co-IP assays were performed to study the binding between TAZ and HBx or HBc; the protein complex was immuno-precipitated with TAZ antibody and further examined by HBx or HBc antibody. Mean±SEM values from at least three independent experiments are presented. * p <0.05, ** p <0.01.

    Journal: Journal of Clinical and Translational Hepatology

    Article Title: RP11-40C6.2 Inactivates Hippo Signaling by Attenuating YAP1 Ubiquitylation in Hepatitis B Virus-associated Hepatocellular Carcinoma

    doi: 10.14218/JCTH.2021.00584

    Figure Lengend Snippet: (A and B) Representative images and analysis of immunohistochemistry staining for YAP1 (×100) and YAP1(s127) (×200) in human HCC subgrouped as shown in the figure. ImageJ was used to analyze the images. (C–E) Linear regression analysis of the expression of RP11-40C6.2 and YAP1 (IOD value) or percentage of YAP1 nuclear translocation and YAP1 (s127) in human HCC. (F) Diagram of potential TEADs binding sites and mutation design within the RP11-40C6.2 promoter region. (G) Luciferase reporter assay of HBx, HBc, and LPA treatment on the activity of wild-type (WT) and TEADs binding site mutation (Mut) RP11-40C6.2 promoter. (H) Antibody to TEAD 1-4 was used in chromatin immunoprecipitation (ChIP) assays; p65 and IgG were negative controls. (I) Co-IP assays were performed to study the binding between TAZ and HBx or HBc; the protein complex was immuno-precipitated with TAZ antibody and further examined by HBx or HBc antibody. Mean±SEM values from at least three independent experiments are presented. * p <0.05, ** p <0.01.

    Article Snippet: Primary polyclonal antibodies targeting anti-YAP1 (#4912; Cell Signaling Technology), anti-YAP1 (s127) (#13008; Cell Signaling Technology), LATS1 (#9153; Cell Signaling Technology), 14-3-3 (#8312; Cell Signaling Technology), anti-HBx (ab203540; Abcam), anti-HBc antibodies (ab8637; Abcam), and anti-TAZ (ab652; Abcam) were used.

    Techniques: Immunohistochemistry, Staining, Expressing, Translocation Assay, Binding Assay, Mutagenesis, Luciferase, Reporter Assay, Activity Assay, Chromatin Immunoprecipitation, Co-Immunoprecipitation Assay

    (A) Real-time PCR in SMMC-7721, MHCC-97H, Huh7, and Hep3B cells shows the expression of RP11-40C6.2. (B) CCK8 assays show the proliferation of SMMC-7721, MHCC-97H, Huh7, and Hep3B cells. (C) Transwell assays show the invasiveness of SMMC-7721, MHCC-97H, Huh7, and Hep3B cells. (D–F) The expression of RP11-40C6.2 and proliferation and invasiveness of Hep3B, SMMC-7721-RP11-40C6.2, Hep3B, and Hep3B-RP11-40C6.2 shRNA. (G, H) Results of CCK8 and Transwell assays of proliferation and invasiveness of SMMC-7721-RP11-40C6.2 and Hep3B cells treated with or without CA3. (I–J) HCC cell line was treated with or without LPA (0.1 µM) and protein expression of YAP1(s127) was assayed by western blotting with beta actin as an internal control. Mean±SEM values from at least three independent experiments are shown. * p <0.05, ** p <0.01.

    Journal: Journal of Clinical and Translational Hepatology

    Article Title: RP11-40C6.2 Inactivates Hippo Signaling by Attenuating YAP1 Ubiquitylation in Hepatitis B Virus-associated Hepatocellular Carcinoma

    doi: 10.14218/JCTH.2021.00584

    Figure Lengend Snippet: (A) Real-time PCR in SMMC-7721, MHCC-97H, Huh7, and Hep3B cells shows the expression of RP11-40C6.2. (B) CCK8 assays show the proliferation of SMMC-7721, MHCC-97H, Huh7, and Hep3B cells. (C) Transwell assays show the invasiveness of SMMC-7721, MHCC-97H, Huh7, and Hep3B cells. (D–F) The expression of RP11-40C6.2 and proliferation and invasiveness of Hep3B, SMMC-7721-RP11-40C6.2, Hep3B, and Hep3B-RP11-40C6.2 shRNA. (G, H) Results of CCK8 and Transwell assays of proliferation and invasiveness of SMMC-7721-RP11-40C6.2 and Hep3B cells treated with or without CA3. (I–J) HCC cell line was treated with or without LPA (0.1 µM) and protein expression of YAP1(s127) was assayed by western blotting with beta actin as an internal control. Mean±SEM values from at least three independent experiments are shown. * p <0.05, ** p <0.01.

    Article Snippet: Primary polyclonal antibodies targeting anti-YAP1 (#4912; Cell Signaling Technology), anti-YAP1 (s127) (#13008; Cell Signaling Technology), LATS1 (#9153; Cell Signaling Technology), 14-3-3 (#8312; Cell Signaling Technology), anti-HBx (ab203540; Abcam), anti-HBc antibodies (ab8637; Abcam), and anti-TAZ (ab652; Abcam) were used.

    Techniques: Real-time Polymerase Chain Reaction, Expressing, shRNA, Western Blot

    (A, B) catRAPID was used to predict hidden binding sites of RP11-40C6.2 and YAP1. The potential binding regions and interaction marks are shown. (C) RNA pull-down performed with sense and antisense probes specific for RP11-40C6.2 and controlled by beads. Western blotting detected precipitated and input total proteins with YAP1 antibody. (D) Ubiquitination detection of YAP1 in SMMC-7721 and Hep3B cells transfected with WT and MU RP11-40C6.2. (E) Proliferation of SMMC-7721 and Hep3B cells transfected with WT and MU RP11-40C6.2. (F) IF staining assay was performed to detect YAP1 nuclear translocation in SMMC-7721 and Hep3B cells transfected with WT and MU RP11-40C6.2. (G) Transwell cell migration assay was used to assess invasion of SMMC-7721 and Hep3B cells transfected with WT and MU RP11-40C6.2. Mean±SEM values from at least three independent experiments are presented. * p <0.05, ** p <0.01.

    Journal: Journal of Clinical and Translational Hepatology

    Article Title: RP11-40C6.2 Inactivates Hippo Signaling by Attenuating YAP1 Ubiquitylation in Hepatitis B Virus-associated Hepatocellular Carcinoma

    doi: 10.14218/JCTH.2021.00584

    Figure Lengend Snippet: (A, B) catRAPID was used to predict hidden binding sites of RP11-40C6.2 and YAP1. The potential binding regions and interaction marks are shown. (C) RNA pull-down performed with sense and antisense probes specific for RP11-40C6.2 and controlled by beads. Western blotting detected precipitated and input total proteins with YAP1 antibody. (D) Ubiquitination detection of YAP1 in SMMC-7721 and Hep3B cells transfected with WT and MU RP11-40C6.2. (E) Proliferation of SMMC-7721 and Hep3B cells transfected with WT and MU RP11-40C6.2. (F) IF staining assay was performed to detect YAP1 nuclear translocation in SMMC-7721 and Hep3B cells transfected with WT and MU RP11-40C6.2. (G) Transwell cell migration assay was used to assess invasion of SMMC-7721 and Hep3B cells transfected with WT and MU RP11-40C6.2. Mean±SEM values from at least three independent experiments are presented. * p <0.05, ** p <0.01.

    Article Snippet: Primary polyclonal antibodies targeting anti-YAP1 (#4912; Cell Signaling Technology), anti-YAP1 (s127) (#13008; Cell Signaling Technology), LATS1 (#9153; Cell Signaling Technology), 14-3-3 (#8312; Cell Signaling Technology), anti-HBx (ab203540; Abcam), anti-HBc antibodies (ab8637; Abcam), and anti-TAZ (ab652; Abcam) were used.

    Techniques: Binding Assay, Western Blot, Transfection, Staining, Translocation Assay, Cell Migration Assay

    Generation of chimeric human/mouse CD79 knockin mice. (A) Schematic representation of B cell Ag receptor complex. (B) CD79 amino acid conservation between mice and humans. Black intervals represent regions of 0% amino acid conservation. Domain demarcation: CY, cytoplasmic; EC, extracellular; L, leader; TM, transmembrane. Conserved cysteines required for interchain disulfide bonding shown for each. (C) Schematic representation of cCD79. Human sequences comprise the CD79B extracellular domain and CD79A extracellular/transmembrane domains. (D) Surface staining of splenic B cells (B220+) from chimeric (h/m)CD79 knockin mice and control C57BL/6 mice. Gray lines show B220−. (E) Rabbit anti-mCD79 immunoblot analysis of whole-cell lysates (purified splenic B cells, CD43−) from cCD79 and WT mice. Upper membrane probed with a polyclonal rabbit Ab raised against a mCD79A and mCD79B extracellular domain fusion protein (Cambier laboratory). Middle membrane probed with a polyclonal rabbit Ab raised against the cytoplasmic domain of mCD79B (Cambier laboratory). An anti–β actin blot was used as a protein loading control. (F) Surface staining of B cell gated (CD19+) human PBMCs with anti-hCD79B (AT-105). Gray line shows CD19−. (G) Surface staining as a function of cCD79B allele dosage. Splenocytes from cCD79 mice of the indicated genotypes were stained with both anti-hCD79B (AT-105) and anti-mCD79B (HM79). Gray contour shows B220−. (H) IgM and IgD surface expression in chimeric mice described in (G). Gray line shows B220−. n = 4 female mice per group for (G) and (H). Error bars represent SEM. All data represent at least three independent experiments; representative data are shown.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Preclinical analysis of candidate anti-human CD79 therapeutic antibodies using a humanized CD79 mouse model

    doi: 10.4049/jimmunol.2101056

    Figure Lengend Snippet: Generation of chimeric human/mouse CD79 knockin mice. (A) Schematic representation of B cell Ag receptor complex. (B) CD79 amino acid conservation between mice and humans. Black intervals represent regions of 0% amino acid conservation. Domain demarcation: CY, cytoplasmic; EC, extracellular; L, leader; TM, transmembrane. Conserved cysteines required for interchain disulfide bonding shown for each. (C) Schematic representation of cCD79. Human sequences comprise the CD79B extracellular domain and CD79A extracellular/transmembrane domains. (D) Surface staining of splenic B cells (B220+) from chimeric (h/m)CD79 knockin mice and control C57BL/6 mice. Gray lines show B220−. (E) Rabbit anti-mCD79 immunoblot analysis of whole-cell lysates (purified splenic B cells, CD43−) from cCD79 and WT mice. Upper membrane probed with a polyclonal rabbit Ab raised against a mCD79A and mCD79B extracellular domain fusion protein (Cambier laboratory). Middle membrane probed with a polyclonal rabbit Ab raised against the cytoplasmic domain of mCD79B (Cambier laboratory). An anti–β actin blot was used as a protein loading control. (F) Surface staining of B cell gated (CD19+) human PBMCs with anti-hCD79B (AT-105). Gray line shows CD19−. (G) Surface staining as a function of cCD79B allele dosage. Splenocytes from cCD79 mice of the indicated genotypes were stained with both anti-hCD79B (AT-105) and anti-mCD79B (HM79). Gray contour shows B220−. (H) IgM and IgD surface expression in chimeric mice described in (G). Gray line shows B220−. n = 4 female mice per group for (G) and (H). Error bars represent SEM. All data represent at least three independent experiments; representative data are shown.

    Article Snippet: The next day primary Abs against intracellular signaling molecules, that is, phosphotyrosine (4G10)-AF647, actin (Santa Cruz, sc1616), p-Syk (Cell Signaling Technology, 2711), pIga (Cell Signaling Technology, 5713), and affinity-purified polyclonal rabbit Abs made in-house, including anti-Syk, mCD79B cytoplasmic tail, and mCD79 extracellular domains, were added in blocking buffer and incubated for 2 h while rocking at RT.

    Techniques: Knock-In, Staining, Western Blot, Purification, Expressing

    BCR signaling and B cell immune responses are unaffected by expression of cCD79. (A) BCR-mediated global tyrosine phosphorylation in chimeric and control B cells. Cell equivalents (2 × 106; CD43−) per lane, resting or stimulated with 10 µg/ml rabbit F(ab′)2 anti-mIg (H+L) for 5 min. Unstimulated cells were run in parallel (left four lanes) to show basal phosphorylation levels. Protein-laden PVDF membranes probed with Abs against p-Tyr (4G10) and actin. (B) BCR-mediated CD79A phosphorylation. Prepared as in (A), probed with anti–p-CD79A (Y182) (rabbit polyclonal anti-mouse pCD79A [Y182]). An anti-mCD79B (see (Fig. 1E), which recognizes the cytoplasmic tail of mCD79B, was used together with actin to normalize the relative abundance of phosphorylated mCD79A (p-CD79A/p-CD79B/actin). Normalized band densities are depicted to the right. (C) BCR-mediated Syk phosphorylation. Prepared as in (A), probed with anti–p-Syk (Y252) (polyclonal rabbit anti–p-Syk [Y525/526]) and biotinylated anti-Syk (in-house) followed by fluorescently conjugated streptavidin. Relative, normalized band densities (pSyk/Syk) are depicted to the right. (D) Representative relative intracellular free calcium before and after BCR stimulation. Splenocytes, stained with anti-B220 and loaded with Indo-1 AM, were stimulated with 1 or 10 µg/ml F(ab′)2 of either goat anti-mIgM (upper traces) or rabbit anti-mIg (H+L) (lower), approximating IgM-only and total BCR stimulation, respectively. Poststimulation, basal-normalized area under the curve (AUC) is depicted on the right. (E) Total (NP19-binding) and high affinity (NP2-binding) IgG+ ELISPOT quantification, 16 d postimmunization with NP-conjugated OVA in alum. (F) Relative serum concentrations of IgG anti-NP Abs from mice immunized in (E) at 16 d postimmunization. NI, not immunized. (G) Total (NP19 binding) IgM+ ELISPOT quantification, at 7 days postimmunization with NP59-Ficoll. n = 3 male and 3 female mice per group. Error bars show SEM. All data represent at least three independent experiments; representative data are shown.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Preclinical analysis of candidate anti-human CD79 therapeutic antibodies using a humanized CD79 mouse model

    doi: 10.4049/jimmunol.2101056

    Figure Lengend Snippet: BCR signaling and B cell immune responses are unaffected by expression of cCD79. (A) BCR-mediated global tyrosine phosphorylation in chimeric and control B cells. Cell equivalents (2 × 106; CD43−) per lane, resting or stimulated with 10 µg/ml rabbit F(ab′)2 anti-mIg (H+L) for 5 min. Unstimulated cells were run in parallel (left four lanes) to show basal phosphorylation levels. Protein-laden PVDF membranes probed with Abs against p-Tyr (4G10) and actin. (B) BCR-mediated CD79A phosphorylation. Prepared as in (A), probed with anti–p-CD79A (Y182) (rabbit polyclonal anti-mouse pCD79A [Y182]). An anti-mCD79B (see (Fig. 1E), which recognizes the cytoplasmic tail of mCD79B, was used together with actin to normalize the relative abundance of phosphorylated mCD79A (p-CD79A/p-CD79B/actin). Normalized band densities are depicted to the right. (C) BCR-mediated Syk phosphorylation. Prepared as in (A), probed with anti–p-Syk (Y252) (polyclonal rabbit anti–p-Syk [Y525/526]) and biotinylated anti-Syk (in-house) followed by fluorescently conjugated streptavidin. Relative, normalized band densities (pSyk/Syk) are depicted to the right. (D) Representative relative intracellular free calcium before and after BCR stimulation. Splenocytes, stained with anti-B220 and loaded with Indo-1 AM, were stimulated with 1 or 10 µg/ml F(ab′)2 of either goat anti-mIgM (upper traces) or rabbit anti-mIg (H+L) (lower), approximating IgM-only and total BCR stimulation, respectively. Poststimulation, basal-normalized area under the curve (AUC) is depicted on the right. (E) Total (NP19-binding) and high affinity (NP2-binding) IgG+ ELISPOT quantification, 16 d postimmunization with NP-conjugated OVA in alum. (F) Relative serum concentrations of IgG anti-NP Abs from mice immunized in (E) at 16 d postimmunization. NI, not immunized. (G) Total (NP19 binding) IgM+ ELISPOT quantification, at 7 days postimmunization with NP59-Ficoll. n = 3 male and 3 female mice per group. Error bars show SEM. All data represent at least three independent experiments; representative data are shown.

    Article Snippet: The next day primary Abs against intracellular signaling molecules, that is, phosphotyrosine (4G10)-AF647, actin (Santa Cruz, sc1616), p-Syk (Cell Signaling Technology, 2711), pIga (Cell Signaling Technology, 5713), and affinity-purified polyclonal rabbit Abs made in-house, including anti-Syk, mCD79B cytoplasmic tail, and mCD79 extracellular domains, were added in blocking buffer and incubated for 2 h while rocking at RT.

    Techniques: Expressing, Staining, Binding Assay, Enzyme-linked Immunospot

    Anti-hCD79A treatment induces suppression of BCR-mediated calcium mobilization. (A) BCR-mediated calcium signaling in B cells from cCD79A mice receiving a 250-µg i.p. injection of anti-hCD79A D265A or control hIgG4 24 h prior. mBCR expression (left) was measured and gated by staining with a polyclonal goat Fab anti-mIgG (H+L). Anti-hCD79A, solid line; control hIgG4, dashed line. Cells were restimulated with 1 or 10 µg/ml rat anti-mIgM (B76). The poststimulation area under the curve (AUC) was normalized to basal calcium levels (right). (B) Cells prepared as in (A) stimulated with 10 µM ionomycin. (C) BCR-mediated calcium signaling in Ramos cells after overnight in vitro incubation with 25 µg/ml hIgG4 anti-hCD79A D265A (solid line) or isotype control hIgG4 (dashed line). Cells were restimulated with either 1 or 10 µg/ml goat F(ab′)2 anti-hIgM Cµ5. (D) Flow cytometric (left and mean fluorescence intensity [MFI] bar graph) and Western blot (right and densitometry) analysis of PTEN expression in B cells from cCD79A animals receiving a 250-µg i.p. injection of either anti-hCD79A D265A or control hIgG4, 24 h prior. For flow cytometry, RBC-lysed splenocytes were fixed and permeabilized before staining with Abs against B220 and PTEN. Gray histogram represents staining isotype control Ab. hIgG4, dashed line; anti-hCD79A D265A, solid line. For Western blot, membranes were prepared as in (Fig. 3D, without BCR stimulation, before being probed with Abs against PTEN or actin. (E) hCD20-CreTAM × ROSA26-STOPflox-YFP × PTENflox/flox or hCD20-CreTAM × ROSA-26-STOPflox-YFP × PTENWT mice were given 2-mg i.p. injections of tamoxifen (TAM) on day 0. On day 7 after TAM, mice were injected i.p. with 0.25 mg of either mIgG2a D265A anti-mCD79B or control mIgG2a anti-HEL. Eighteen hours after Ab injection, B220+YFP+ splenocytes were analyzed by flow for PTEN expression and Ab coating. (F) As before, calcium was measured as a function of equal mBCR expression. n = 3 mice per group. Error bars show SEM. All data represents at least three independent experiments; representative data are shown. A Student t test was used to evaluate statistical significance. *p < 0.05, **p < 0.01, ****p < 0.0001.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Preclinical analysis of candidate anti-human CD79 therapeutic antibodies using a humanized CD79 mouse model

    doi: 10.4049/jimmunol.2101056

    Figure Lengend Snippet: Anti-hCD79A treatment induces suppression of BCR-mediated calcium mobilization. (A) BCR-mediated calcium signaling in B cells from cCD79A mice receiving a 250-µg i.p. injection of anti-hCD79A D265A or control hIgG4 24 h prior. mBCR expression (left) was measured and gated by staining with a polyclonal goat Fab anti-mIgG (H+L). Anti-hCD79A, solid line; control hIgG4, dashed line. Cells were restimulated with 1 or 10 µg/ml rat anti-mIgM (B76). The poststimulation area under the curve (AUC) was normalized to basal calcium levels (right). (B) Cells prepared as in (A) stimulated with 10 µM ionomycin. (C) BCR-mediated calcium signaling in Ramos cells after overnight in vitro incubation with 25 µg/ml hIgG4 anti-hCD79A D265A (solid line) or isotype control hIgG4 (dashed line). Cells were restimulated with either 1 or 10 µg/ml goat F(ab′)2 anti-hIgM Cµ5. (D) Flow cytometric (left and mean fluorescence intensity [MFI] bar graph) and Western blot (right and densitometry) analysis of PTEN expression in B cells from cCD79A animals receiving a 250-µg i.p. injection of either anti-hCD79A D265A or control hIgG4, 24 h prior. For flow cytometry, RBC-lysed splenocytes were fixed and permeabilized before staining with Abs against B220 and PTEN. Gray histogram represents staining isotype control Ab. hIgG4, dashed line; anti-hCD79A D265A, solid line. For Western blot, membranes were prepared as in (Fig. 3D, without BCR stimulation, before being probed with Abs against PTEN or actin. (E) hCD20-CreTAM × ROSA26-STOPflox-YFP × PTENflox/flox or hCD20-CreTAM × ROSA-26-STOPflox-YFP × PTENWT mice were given 2-mg i.p. injections of tamoxifen (TAM) on day 0. On day 7 after TAM, mice were injected i.p. with 0.25 mg of either mIgG2a D265A anti-mCD79B or control mIgG2a anti-HEL. Eighteen hours after Ab injection, B220+YFP+ splenocytes were analyzed by flow for PTEN expression and Ab coating. (F) As before, calcium was measured as a function of equal mBCR expression. n = 3 mice per group. Error bars show SEM. All data represents at least three independent experiments; representative data are shown. A Student t test was used to evaluate statistical significance. *p < 0.05, **p < 0.01, ****p < 0.0001.

    Article Snippet: The next day primary Abs against intracellular signaling molecules, that is, phosphotyrosine (4G10)-AF647, actin (Santa Cruz, sc1616), p-Syk (Cell Signaling Technology, 2711), pIga (Cell Signaling Technology, 5713), and affinity-purified polyclonal rabbit Abs made in-house, including anti-Syk, mCD79B cytoplasmic tail, and mCD79 extracellular domains, were added in blocking buffer and incubated for 2 h while rocking at RT.

    Techniques: Injection, Expressing, Staining, In Vitro, Incubation, Fluorescence, Western Blot, Flow Cytometry

    ( A ) Schematic representation of GFP-TACC3 (Δ678–681)-TACC3 shRNA and GFP-TACC3 (Δ682–688)-TACC3 shRNA constructs. ( B ) Lysates of HeLa Kyoto cells transfected with GFP-TACC3 (WT)-TACC3 shRNA, GFP-TACC3 (Δ682–688)-TACC3 shRNA, or GFP-TACC3 (Δ678–681)-TACC3 shRNA for 48 h were analyzed for exogenous TACC3 proteins with simultaneous depletion of endogenous TACC3. Both endogenous TACC3 and GFP-TACC3 proteins were stained with mouse monoclonal TACC3 antibody. Actin was probed as a control. TACC3 level in control untrasfected cells is also shown. ( C ) Immunoprecipitation of GFP-tagged TACC3 proteins using the anti-GFP antibody in the lysates of HeLa Kyoto cells transfected with GFP-TACC3 (WT)-TACC3 shRNA, GFP-TACC3 (Δ678–681)-TACC3 shRNA, or GFP-TACC3 (Δ682–688)-TACC3 shRNA. The immunoblots were probed with GFP and ch-TOG antibodies. ( D ) Representative confocal images of GFP-TACC3 (WT)-TACC3 shRNA (top panel), GFP-TACC3 (Δ678-681)-TACC3 shRNA (middle panel), or GFP-TACC3 (Δ682–688)-TACC3 shRNA (bottom panel)- transfected mitotic HeLa Kyoto cells showing the differences in γ-tubulin levels at the centrosomes. Scale bar, 5 μm, γ-tubulin, and ch-TOG were stained with mouse monoclonal anti-γ-tubulin and rabbit polyclonal anti-ch-TOG antibody, respectively. ( E ) The bar graph shows the quantification of γ-tubulin intensity at the centrosomes (two) in different conditions as of (d). ( F ) Representative confocal images of GFP-TACC3 (WT)-TACC3 shRNA (top panel), GFP-TACC3 (Δ678–681)-TACC3 shRNA (middle panel), or GFP-TACC3 (Δ682–688)-TACC3 shRNA (bottom panel) transfected mitotic HeLa Kyoto cells showing the differences in GCP6 localization at centrosomes. Scale bar, 5 μm, GCP6 was stained with mouse monoclonal anti-GCP6 antibody. ( G ) The bar graph shows the quantification of GCP6 intensity at the centrosomes in different conditions as in panel (F). ( H ) The bar graph shows the percentage of cells (cells with above the average centrosomal γ-tubulin intensity of TACC3 WT-expressed cells) with increased γ-tubulin intensity phenotype in both the mutant cases. The bars in (e), (g), and (h) represent mean ± S.E. The number of mitotic cells counted = 100 each (four independent experiments); ****, P <0.0001, ***, P <0.001.

    Journal: Bioscience Reports

    Article Title: TACC3–ch-TOG interaction regulates spindle microtubule assembly by controlling centrosomal recruitment of γ-TuRC

    doi: 10.1042/BSR20221882

    Figure Lengend Snippet: ( A ) Schematic representation of GFP-TACC3 (Δ678–681)-TACC3 shRNA and GFP-TACC3 (Δ682–688)-TACC3 shRNA constructs. ( B ) Lysates of HeLa Kyoto cells transfected with GFP-TACC3 (WT)-TACC3 shRNA, GFP-TACC3 (Δ682–688)-TACC3 shRNA, or GFP-TACC3 (Δ678–681)-TACC3 shRNA for 48 h were analyzed for exogenous TACC3 proteins with simultaneous depletion of endogenous TACC3. Both endogenous TACC3 and GFP-TACC3 proteins were stained with mouse monoclonal TACC3 antibody. Actin was probed as a control. TACC3 level in control untrasfected cells is also shown. ( C ) Immunoprecipitation of GFP-tagged TACC3 proteins using the anti-GFP antibody in the lysates of HeLa Kyoto cells transfected with GFP-TACC3 (WT)-TACC3 shRNA, GFP-TACC3 (Δ678–681)-TACC3 shRNA, or GFP-TACC3 (Δ682–688)-TACC3 shRNA. The immunoblots were probed with GFP and ch-TOG antibodies. ( D ) Representative confocal images of GFP-TACC3 (WT)-TACC3 shRNA (top panel), GFP-TACC3 (Δ678-681)-TACC3 shRNA (middle panel), or GFP-TACC3 (Δ682–688)-TACC3 shRNA (bottom panel)- transfected mitotic HeLa Kyoto cells showing the differences in γ-tubulin levels at the centrosomes. Scale bar, 5 μm, γ-tubulin, and ch-TOG were stained with mouse monoclonal anti-γ-tubulin and rabbit polyclonal anti-ch-TOG antibody, respectively. ( E ) The bar graph shows the quantification of γ-tubulin intensity at the centrosomes (two) in different conditions as of (d). ( F ) Representative confocal images of GFP-TACC3 (WT)-TACC3 shRNA (top panel), GFP-TACC3 (Δ678–681)-TACC3 shRNA (middle panel), or GFP-TACC3 (Δ682–688)-TACC3 shRNA (bottom panel) transfected mitotic HeLa Kyoto cells showing the differences in GCP6 localization at centrosomes. Scale bar, 5 μm, GCP6 was stained with mouse monoclonal anti-GCP6 antibody. ( G ) The bar graph shows the quantification of GCP6 intensity at the centrosomes in different conditions as in panel (F). ( H ) The bar graph shows the percentage of cells (cells with above the average centrosomal γ-tubulin intensity of TACC3 WT-expressed cells) with increased γ-tubulin intensity phenotype in both the mutant cases. The bars in (e), (g), and (h) represent mean ± S.E. The number of mitotic cells counted = 100 each (four independent experiments); ****, P <0.0001, ***, P <0.001.

    Article Snippet: Ser 558-phosphorylated TACC3 was probed by rabbit polyclonal phospho-TACC3 antibody (Cell Signaling, U.S.A.).

    Techniques: shRNA, Construct, Transfection, Staining, Immunoprecipitation, Western Blot, Mutagenesis

    ( A ) Representative confocal images of GFP-TACC3 (WT)-TACC3 shRNA (top panel), GFP-TACC3 (Δ678–681)-TACC3 shRNA (middle panel), or GFP-TACC3 (Δ682–688)-TACC3 shRNA (bottom panel) transfected mitotic HeLa Kyoto cells showing the differences in spindle microtubule density. Scale bar, 5 μm, α-tubulin, and ch-TOG were stained with mouse monoclonal anti-α-tubulin and rabbit polyclonal anti-ch-TOG, respectively. ( B ) The bar graph shows the quantification of α-tubulin/MT intensity at the centrosomes (two). ( C ) The bar graph shows the percentage of cells (cells with above the average centrosomal microtubule intensity of TACC3 WT-expressed cells) with increased microtubule density phenotype in both the mutant cases. ( D ) The bar graph shows the quantification of ch-TOG intensity at the centrosomes ( E ) The bar graph shows the quantification of GFP-TACC3 intensity at the centrosomes. ( F ) The bar graph shows the quantification of ch-TOG intensity on the mitotic spindles. The regions of interest (ROI) used for quantification are shown in panel (A). ( G ) The bar graph shows the percentage of cells (cells with above the average ch-TOG intensity of TACC3 WT-expressed cells) with decreased ch-TOG intensity at the centrosomes phenotype in both the mutant cases. ( H ) The bar graph shows the percentage of cells (cells with above the average ch-TOG intensity of TACC3 WT-expressed cells) with increased ch-TOG intensity on the spindles in both the mutant cases. The bars represent mean ± S.E. The number of mitotic cells counted = 90–100 each (four independent experiments). ****, P <0.0001, ***, P < 0.001, **, P <0.01, *, P <0.05.

    Journal: Bioscience Reports

    Article Title: TACC3–ch-TOG interaction regulates spindle microtubule assembly by controlling centrosomal recruitment of γ-TuRC

    doi: 10.1042/BSR20221882

    Figure Lengend Snippet: ( A ) Representative confocal images of GFP-TACC3 (WT)-TACC3 shRNA (top panel), GFP-TACC3 (Δ678–681)-TACC3 shRNA (middle panel), or GFP-TACC3 (Δ682–688)-TACC3 shRNA (bottom panel) transfected mitotic HeLa Kyoto cells showing the differences in spindle microtubule density. Scale bar, 5 μm, α-tubulin, and ch-TOG were stained with mouse monoclonal anti-α-tubulin and rabbit polyclonal anti-ch-TOG, respectively. ( B ) The bar graph shows the quantification of α-tubulin/MT intensity at the centrosomes (two). ( C ) The bar graph shows the percentage of cells (cells with above the average centrosomal microtubule intensity of TACC3 WT-expressed cells) with increased microtubule density phenotype in both the mutant cases. ( D ) The bar graph shows the quantification of ch-TOG intensity at the centrosomes ( E ) The bar graph shows the quantification of GFP-TACC3 intensity at the centrosomes. ( F ) The bar graph shows the quantification of ch-TOG intensity on the mitotic spindles. The regions of interest (ROI) used for quantification are shown in panel (A). ( G ) The bar graph shows the percentage of cells (cells with above the average ch-TOG intensity of TACC3 WT-expressed cells) with decreased ch-TOG intensity at the centrosomes phenotype in both the mutant cases. ( H ) The bar graph shows the percentage of cells (cells with above the average ch-TOG intensity of TACC3 WT-expressed cells) with increased ch-TOG intensity on the spindles in both the mutant cases. The bars represent mean ± S.E. The number of mitotic cells counted = 90–100 each (four independent experiments). ****, P <0.0001, ***, P < 0.001, **, P <0.01, *, P <0.05.

    Article Snippet: Ser 558-phosphorylated TACC3 was probed by rabbit polyclonal phospho-TACC3 antibody (Cell Signaling, U.S.A.).

    Techniques: shRNA, Transfection, Staining, Mutagenesis

    ( A ) Immunoprecipitation of GFP-tagged TACC3 proteins using GFP antibody in lysates of HeLa Kyoto cells transfected with GFP-TACC3 (WT)-TACC3 shRNA, GFP-TACC3 (Δ678–681)-TACC3 shRNA, or GFP-TACC3 (Δ682–688)-TACC3 shRNA. The immunoblots were probed with GFP-TACC3 (WT), GFP-TACC3 (Δ678–681), and GFP-TACC3 (Δ682–688) forms along with γ-tubulin, GCP3, GCP4, and GCP6 by using the respective antibodies. ( B ) Fold change of different γ-TuRC proteins from the GFP-TACC3 immunoprecipitates of GFP-TACC3 WT, GFP-TACC3 (Δ678–681), or GFP-TACC3 (Δ682–688) expressed cells are plotted (based on three experiments in each). Data are mean ± S.E. ****, P <0.0001, ***, P <0.001, **, P <0.01, ns: not significant.

    Journal: Bioscience Reports

    Article Title: TACC3–ch-TOG interaction regulates spindle microtubule assembly by controlling centrosomal recruitment of γ-TuRC

    doi: 10.1042/BSR20221882

    Figure Lengend Snippet: ( A ) Immunoprecipitation of GFP-tagged TACC3 proteins using GFP antibody in lysates of HeLa Kyoto cells transfected with GFP-TACC3 (WT)-TACC3 shRNA, GFP-TACC3 (Δ678–681)-TACC3 shRNA, or GFP-TACC3 (Δ682–688)-TACC3 shRNA. The immunoblots were probed with GFP-TACC3 (WT), GFP-TACC3 (Δ678–681), and GFP-TACC3 (Δ682–688) forms along with γ-tubulin, GCP3, GCP4, and GCP6 by using the respective antibodies. ( B ) Fold change of different γ-TuRC proteins from the GFP-TACC3 immunoprecipitates of GFP-TACC3 WT, GFP-TACC3 (Δ678–681), or GFP-TACC3 (Δ682–688) expressed cells are plotted (based on three experiments in each). Data are mean ± S.E. ****, P <0.0001, ***, P <0.001, **, P <0.01, ns: not significant.

    Article Snippet: Ser 558-phosphorylated TACC3 was probed by rabbit polyclonal phospho-TACC3 antibody (Cell Signaling, U.S.A.).

    Techniques: Immunoprecipitation, Transfection, shRNA, Western Blot

    ( A ) Schematic representation of GFP-TACC3 (WT) and GFP-TACC3 ΔC (1–590) constructs. Position of Ser 558 phosphorylation site is also shown. ( B ) Lysates of HeLa Kyoto cells transfected with GFP-TACC3 (WT) along with TACC3 3′-UTR siRNA and GFP-TACC3 ΔC (1–590) along with TACC3 esiRNA for >48 h were analyzed by Western blot to detect the levels of exogenous TACC3 proteins and the depletion of endogenous TACC3 by the respective siRNAs. Both endogenous TACC3 and the exogenous TACC3 proteins were probed with mouse monoclonal anti-TACC3 antibody. α-tubulin was probed as a control. ( C ) Representative confocal images of GFP-TACC3 (WT) and GFP-TACC3 ΔC (1–590) transfected mitotic HeLa Kyoto cells showing the differences in γ-tubulin intensity. Scale bar, 5 μm, ( D ) The plots show γ-tubulin intensity at the centrosomes in different conditions as indicated. Centrosomal ROI used for quantification for both cases is shown. ( E ) The bar graph shows the percentage of cells (cells with above the average centrosomal γ-tubulin intensity of TACC3 WT-expressed cells) with decreased γ-tubulin intensity phenotype in GFP-TACC3 ΔC cells. The bars represent mean ± S.E. The number of mitotic cells counted = 60 each (four independent experiments). ****, P <0.0001, ***, P <0.001. ( F ) Immunoprecipitation of GFP-tagged TACC3 ΔC using the anti-GFP antibody in the lysate of HEK293T cells transfected with GFP-TACC3 ΔC. The immunoblots were probed with GFP-TACC3 ΔC, γ-tubulin, GCP3, GCP4, and GCP6 using the respective antibodies. ( G ) Immunoprecipitation of γ-tubulin using anti-γ-tubulin antibody in the lysate of HEK cells transfected with GFP-TACC3 ΔC. The immunoblots were probed with GFP-TACC3 ΔC and γ-tubulin using the respective antibodies. ( H ) Immunoprecipitation of GCP4 using anti-GCP4 antibody in the lysate of HEK293T cells transfected with GFP-TACC3 ΔC. The immunoblots were probed for GFP-TACC3 ΔC along with GCP4 by using respective antibodies.

    Journal: Bioscience Reports

    Article Title: TACC3–ch-TOG interaction regulates spindle microtubule assembly by controlling centrosomal recruitment of γ-TuRC

    doi: 10.1042/BSR20221882

    Figure Lengend Snippet: ( A ) Schematic representation of GFP-TACC3 (WT) and GFP-TACC3 ΔC (1–590) constructs. Position of Ser 558 phosphorylation site is also shown. ( B ) Lysates of HeLa Kyoto cells transfected with GFP-TACC3 (WT) along with TACC3 3′-UTR siRNA and GFP-TACC3 ΔC (1–590) along with TACC3 esiRNA for >48 h were analyzed by Western blot to detect the levels of exogenous TACC3 proteins and the depletion of endogenous TACC3 by the respective siRNAs. Both endogenous TACC3 and the exogenous TACC3 proteins were probed with mouse monoclonal anti-TACC3 antibody. α-tubulin was probed as a control. ( C ) Representative confocal images of GFP-TACC3 (WT) and GFP-TACC3 ΔC (1–590) transfected mitotic HeLa Kyoto cells showing the differences in γ-tubulin intensity. Scale bar, 5 μm, ( D ) The plots show γ-tubulin intensity at the centrosomes in different conditions as indicated. Centrosomal ROI used for quantification for both cases is shown. ( E ) The bar graph shows the percentage of cells (cells with above the average centrosomal γ-tubulin intensity of TACC3 WT-expressed cells) with decreased γ-tubulin intensity phenotype in GFP-TACC3 ΔC cells. The bars represent mean ± S.E. The number of mitotic cells counted = 60 each (four independent experiments). ****, P <0.0001, ***, P <0.001. ( F ) Immunoprecipitation of GFP-tagged TACC3 ΔC using the anti-GFP antibody in the lysate of HEK293T cells transfected with GFP-TACC3 ΔC. The immunoblots were probed with GFP-TACC3 ΔC, γ-tubulin, GCP3, GCP4, and GCP6 using the respective antibodies. ( G ) Immunoprecipitation of γ-tubulin using anti-γ-tubulin antibody in the lysate of HEK cells transfected with GFP-TACC3 ΔC. The immunoblots were probed with GFP-TACC3 ΔC and γ-tubulin using the respective antibodies. ( H ) Immunoprecipitation of GCP4 using anti-GCP4 antibody in the lysate of HEK293T cells transfected with GFP-TACC3 ΔC. The immunoblots were probed for GFP-TACC3 ΔC along with GCP4 by using respective antibodies.

    Article Snippet: Ser 558-phosphorylated TACC3 was probed by rabbit polyclonal phospho-TACC3 antibody (Cell Signaling, U.S.A.).

    Techniques: Construct, Transfection, esiRNA, Western Blot, Immunoprecipitation

    ( A ) Lysates of HeLa Kyoto cells transfected with GFP-TACC3 (WT)-TACC3 shRNA, GFP-TACC3 (Δ682–688)-TACC3 shRNA, or GFP-TACC3 (Δ678–681)-TACC3 shRNA for 48 h were analyzed by Western blot to detect the levels of GFP-TACC3 proteins with simultaneously probing for endogenous TACC3. Both endogenous TACC3 and GFP-TACC3 proteins were probed with mouse monoclonal anti-TACC3 antibody. Phospho-Ser 558 TACC3 was detected using rabbit monoclonal anti-phospho-Ser 558 TACC3 antibody. α-tubulin was probed as a control. ( B ) Representative confocal images of GFP-TACC3 (WT)-TACC3 shRNA (top panel), GFP-TACC3 (Δ678–681)-TACC3 shRNA (middle panel), or GFP-TACC3 (Δ682–688)-TACC3 shRNA (bottom panel)-transfected mitotic HeLa Kyoto cells showing the localization of phospho-Ser 558 TACC3. Scale bar, 5 μm. Phospho-Ser 558 TACC3 was detected using rabbit monoclonal anti-phospho-Ser 558 TACC3 antibody. ( C ) The bar graph shows intensity of phospho-Ser 558 TACC3 at centrosomes in synchronized metaphase cells in ROI (19.47 μm 2 ), as shown, around the centrosomes in different conditions as in panel (B). ( D ) The bar graph shows the percentage of cells (cells with above the average centrosomal phospho-Ser 558 TACC3 intensity of TACC3 WT-expressed cells) showing increased phospho-Ser 558 TACC3 intensity at the centrosomes in both the mutant cases. ( E ) The bar graph shows the quantification of phospho-Ser 558 TACC3 intensity on the mitotic spindles.The ROI used for quantification is shown in (b). ( F ) The bar graph shows the percentage of cells (cells with above the average phospho-Ser 558 TACC3 intensity of TACC3 WT-expressed cells) showing decreased phospho-Ser 558 TACC3 intensity on the spindles in both the mutant cases. The bars represent mean ± S.E. The number of cells counted = 70 each (four independent experiments) ****, P <0.0001, ***, P <0.001.

    Journal: Bioscience Reports

    Article Title: TACC3–ch-TOG interaction regulates spindle microtubule assembly by controlling centrosomal recruitment of γ-TuRC

    doi: 10.1042/BSR20221882

    Figure Lengend Snippet: ( A ) Lysates of HeLa Kyoto cells transfected with GFP-TACC3 (WT)-TACC3 shRNA, GFP-TACC3 (Δ682–688)-TACC3 shRNA, or GFP-TACC3 (Δ678–681)-TACC3 shRNA for 48 h were analyzed by Western blot to detect the levels of GFP-TACC3 proteins with simultaneously probing for endogenous TACC3. Both endogenous TACC3 and GFP-TACC3 proteins were probed with mouse monoclonal anti-TACC3 antibody. Phospho-Ser 558 TACC3 was detected using rabbit monoclonal anti-phospho-Ser 558 TACC3 antibody. α-tubulin was probed as a control. ( B ) Representative confocal images of GFP-TACC3 (WT)-TACC3 shRNA (top panel), GFP-TACC3 (Δ678–681)-TACC3 shRNA (middle panel), or GFP-TACC3 (Δ682–688)-TACC3 shRNA (bottom panel)-transfected mitotic HeLa Kyoto cells showing the localization of phospho-Ser 558 TACC3. Scale bar, 5 μm. Phospho-Ser 558 TACC3 was detected using rabbit monoclonal anti-phospho-Ser 558 TACC3 antibody. ( C ) The bar graph shows intensity of phospho-Ser 558 TACC3 at centrosomes in synchronized metaphase cells in ROI (19.47 μm 2 ), as shown, around the centrosomes in different conditions as in panel (B). ( D ) The bar graph shows the percentage of cells (cells with above the average centrosomal phospho-Ser 558 TACC3 intensity of TACC3 WT-expressed cells) showing increased phospho-Ser 558 TACC3 intensity at the centrosomes in both the mutant cases. ( E ) The bar graph shows the quantification of phospho-Ser 558 TACC3 intensity on the mitotic spindles.The ROI used for quantification is shown in (b). ( F ) The bar graph shows the percentage of cells (cells with above the average phospho-Ser 558 TACC3 intensity of TACC3 WT-expressed cells) showing decreased phospho-Ser 558 TACC3 intensity on the spindles in both the mutant cases. The bars represent mean ± S.E. The number of cells counted = 70 each (four independent experiments) ****, P <0.0001, ***, P <0.001.

    Article Snippet: Ser 558-phosphorylated TACC3 was probed by rabbit polyclonal phospho-TACC3 antibody (Cell Signaling, U.S.A.).

    Techniques: Transfection, shRNA, Western Blot, Mutagenesis

    ( A ) Lysates of HeLa Kyoto cells transfected with control siRNA and ch-TOG siRNA for 48 h were analyzed by Western blot to detect ch-TOG expression. ( B ) Representative confocal images of control siRNA or ch-TOG siRNA transfected mitotic Hela Kyoto cells showing the γ-tubulin intensity at the centrosomes and mitotic spindle. Scale bar, 5 μm. γ-tubulin was stained with mouse monoclonal anti-γ-tubulin antibody. Centrosomal regions are shown in an enlarged view. ( C ) The bar graph shows the quantification of centrosomal γ-tubulin intensity as of (b). The bars represent mean ± S.E. The number of cells counted = 50 each (four independent experiments). ( D ) The bar graph shows the quantification of γ-tubulin intensity on the spindles in different conditions as of (b). ( E ) The bar graph shows the percentage of cells (cells with above the average γ-tubulin intensity of TACC3 WT-expressed cells) showing increased γ-tubulin intensity at the spindles phenotype in ch-TOG siRNA treated cells. The bars represent mean ± S.E. The number of cells counted = 50 each (four independent experiments) ****, P <0.0001, ***, P <0.001. ( F ) Immunoprecipitation of GFP tagged ch-TOG using GFP coated beads (GFP-Trap) in the lysates of HeLa Kyoto cells stably expressing GFP-ch-TOG or control HeLa cells transfected with an empty GFP-vector (pcDNA3-EGFP). The immunoblots were probed for GFP-ch-TOG, γ-tubulin, GCP3, GCP4, GCP6, and TACC3 using the respective antibodies. ( G ) Immunoprecipitation of GFP-tagged ch-TOG C-terminus (1428–2032) using anti-GFP antibody in the lysates of HEK cells transfected with GFP-ch-TOG C-ter. The immunoblots were probed for GFP-ch-TOG, γ-tubulin, GCP3, GCP4, and TACC3 using the respective antibodies.

    Journal: Bioscience Reports

    Article Title: TACC3–ch-TOG interaction regulates spindle microtubule assembly by controlling centrosomal recruitment of γ-TuRC

    doi: 10.1042/BSR20221882

    Figure Lengend Snippet: ( A ) Lysates of HeLa Kyoto cells transfected with control siRNA and ch-TOG siRNA for 48 h were analyzed by Western blot to detect ch-TOG expression. ( B ) Representative confocal images of control siRNA or ch-TOG siRNA transfected mitotic Hela Kyoto cells showing the γ-tubulin intensity at the centrosomes and mitotic spindle. Scale bar, 5 μm. γ-tubulin was stained with mouse monoclonal anti-γ-tubulin antibody. Centrosomal regions are shown in an enlarged view. ( C ) The bar graph shows the quantification of centrosomal γ-tubulin intensity as of (b). The bars represent mean ± S.E. The number of cells counted = 50 each (four independent experiments). ( D ) The bar graph shows the quantification of γ-tubulin intensity on the spindles in different conditions as of (b). ( E ) The bar graph shows the percentage of cells (cells with above the average γ-tubulin intensity of TACC3 WT-expressed cells) showing increased γ-tubulin intensity at the spindles phenotype in ch-TOG siRNA treated cells. The bars represent mean ± S.E. The number of cells counted = 50 each (four independent experiments) ****, P <0.0001, ***, P <0.001. ( F ) Immunoprecipitation of GFP tagged ch-TOG using GFP coated beads (GFP-Trap) in the lysates of HeLa Kyoto cells stably expressing GFP-ch-TOG or control HeLa cells transfected with an empty GFP-vector (pcDNA3-EGFP). The immunoblots were probed for GFP-ch-TOG, γ-tubulin, GCP3, GCP4, GCP6, and TACC3 using the respective antibodies. ( G ) Immunoprecipitation of GFP-tagged ch-TOG C-terminus (1428–2032) using anti-GFP antibody in the lysates of HEK cells transfected with GFP-ch-TOG C-ter. The immunoblots were probed for GFP-ch-TOG, γ-tubulin, GCP3, GCP4, and TACC3 using the respective antibodies.

    Article Snippet: Ser 558-phosphorylated TACC3 was probed by rabbit polyclonal phospho-TACC3 antibody (Cell Signaling, U.S.A.).

    Techniques: Transfection, Western Blot, Expressing, Staining, Immunoprecipitation, Stable Transfection, Plasmid Preparation

    (A) Western blotting and Coomassie staining of GFP-IPs from MCF10A GFP-LC3A expressing cells treated +/- LLOMe (250 µM, 40 mins). (B) Normalized mass spectrometry analysis of LC3A-PE and LC3A-PS in cells treated as in (A). (C) Representative confocal images of GFP-LC3A in wild type and ATG13 KO MCF10A cells treated with LLOMe (250 µM, 30 min) or PP242 (1 µM, 1 hr), after pre-treatment with BafA1 (100 nM). Scale bar, 10 µm. (D) Representative confocal images of endogenous LC3 in wild type MCF10A cells treated as in (C). Scale bar, 10 µm. (E) Confocal images of wild type or ATG13 KO MCF10A cells expressing GFP-LC3A treated with LLOMe (250 µM, 20 min) +/- BafA1 pre-treatment (100 nM) and stained for Galectin-3. Inserts show single channel cropped images. Scale bar, 5 µm. MCF10A cells were treated with monensin (100 μM), SaliP (2.5 μM), PP242 (1 µM), LLOMe (250 μM) or LLOMe + BafA1 (100 nM) for 1 h. Following fractionation, membrane and cytosol fractions were probed for ATP6V1A, ATP6V0D1 by western blotting. V1/V0 ratios shown below. (G) Western blotting analysis of GFP-LC3A expressing wild type MCF10A and ATG16L1 KO cells re-expressing ATG16L1 WT or K490A, treated with LLOMe (250 µM, 20 min). (H) Confocal images of GFP-LC3A in the MCF10A ATG16L1 cell line panel treated with LLOMe (250 µM, 20 mins) or MSU crystals (200 µg/ml, 4 hr) and co stained for LAMP1. Arrows denote LAMP1 positive crystals. Scale bar, 10 µm. (I) Confocal images of GFP-LC3A expressing MCF10A cells co-transfected with mCherry-SopF. Outlined cells mark mCherry-SopF expressing cells. Scale bar, 20 µm.

    Journal: bioRxiv

    Article Title: Lysosome damage triggers direct ATG8 conjugation and ATG2 engagement via CASM

    doi: 10.1101/2023.03.22.533754

    Figure Lengend Snippet: (A) Western blotting and Coomassie staining of GFP-IPs from MCF10A GFP-LC3A expressing cells treated +/- LLOMe (250 µM, 40 mins). (B) Normalized mass spectrometry analysis of LC3A-PE and LC3A-PS in cells treated as in (A). (C) Representative confocal images of GFP-LC3A in wild type and ATG13 KO MCF10A cells treated with LLOMe (250 µM, 30 min) or PP242 (1 µM, 1 hr), after pre-treatment with BafA1 (100 nM). Scale bar, 10 µm. (D) Representative confocal images of endogenous LC3 in wild type MCF10A cells treated as in (C). Scale bar, 10 µm. (E) Confocal images of wild type or ATG13 KO MCF10A cells expressing GFP-LC3A treated with LLOMe (250 µM, 20 min) +/- BafA1 pre-treatment (100 nM) and stained for Galectin-3. Inserts show single channel cropped images. Scale bar, 5 µm. MCF10A cells were treated with monensin (100 μM), SaliP (2.5 μM), PP242 (1 µM), LLOMe (250 μM) or LLOMe + BafA1 (100 nM) for 1 h. Following fractionation, membrane and cytosol fractions were probed for ATP6V1A, ATP6V0D1 by western blotting. V1/V0 ratios shown below. (G) Western blotting analysis of GFP-LC3A expressing wild type MCF10A and ATG16L1 KO cells re-expressing ATG16L1 WT or K490A, treated with LLOMe (250 µM, 20 min). (H) Confocal images of GFP-LC3A in the MCF10A ATG16L1 cell line panel treated with LLOMe (250 µM, 20 mins) or MSU crystals (200 µg/ml, 4 hr) and co stained for LAMP1. Arrows denote LAMP1 positive crystals. Scale bar, 10 µm. (I) Confocal images of GFP-LC3A expressing MCF10A cells co-transfected with mCherry-SopF. Outlined cells mark mCherry-SopF expressing cells. Scale bar, 20 µm.

    Article Snippet: Antibodies used were rabbit polyclonal anti-ATG16L1 (8090, Cell Signalling, WB 1:1000), rabbit polyclonal anti-LC3A/B (4108, Cell Signalling, WB 1:1000, IF 1:100), rabbit monoclonal anti-ATP6V1A (ab199326, Abcam, WB 1:2000), mouse monoclonal anti-ATP6V0d1 (ab56441, Abcam, WB 1:1000), mouse monoclonal anti-LAMP1 (555798, BD Biosciences, IF 1:100), mouse monoclonal anti LAMP1 (611042, BD Bioscience, WB, 1:500), mouse monoclonal anti-GAPDH (ab8245, Abcam, WB 1:1000), rabbit anti-ATG13 (6940, Cell Signalling, WB 1:1000), mouse anti-GFP (1181446000, Roche, WB 1:1000), mouse anti-WIPI2 (MCA5780GA, BioRad, IF 1:100), rabbit anti ATG2B (25155-1-AP, ProteinTech, WB 1:1000), mouse anti-Galectin 3 (32790, Scbt, IF 1:100), rabbit anti-b tubulin (2128, Cell Signalling, WB 1:1000), Alexa Fluor 488 polyclonal goat anti-rabbit IgG (A-11034, ThermoFisher, IF 1:500), Alexa Fluor 568 polyclonal goat anti-mouse IgG (A-11004, ThermoFisher, IF 1:500), Alexa Fluor 568 polyclonal goat anti-rabbit IgG (A-11011, ThermoFisher, IF 1:500), HRP-conjugated anti-rabbit IgG (7074, Cell Signalling, WB 1:2000), HRP-conjugated anti-mouse IgG (7076, Cell Signalling, WB 1:2000).

    Techniques: Western Blot, Staining, Expressing, Mass Spectrometry, Fractionation, Transfection

    (A) Western blot analysis of GFP-IPs and input from wild type and ATG13 KO MCF10A cells expressing either GFP or GFP-LC3A treated +/- LLOMe (250 µM, 30 mins). Samples probed for ATG2B, GFP, ATG13 and GAPDH. (B) Western blot analysis of GFP-IPs and input from MCF10A ATG16L1 cell line panel expressing GFP-LC3A treated +/- LLOMe (250 µM, 30 mins). Samples probed for ATG2B, GFP, ATG16L1 and GAPDH. (C) Western blot analysis of GFP-IPs and total cell lysate (TCL) from GFP-LC3A expressing MCF10A cells treated with LLOMe (250 µM, 30 mins) or PP242 (1 µM, 1 hr) +/- Vps34 IN-1 (5 µM). Samples probed for ATG2B, GFP and GAPDH. (D) GFP-LC3A expressing ATG13 or ATG16L1 KO MCF10A cells were treated with LLOMe (250 µM, 30 mins). Following fractionation, input, cytosol and membrane fractions were probed for ATG2B, LAMP1, GFP and β-tubulin. (E) Western blot analysis of GFP-IPs and input from ATG13 KO MCF10A cells expressing GFP-LC3A treated with LLOMe (250 µM, 30mins), GPN (200 µM, 30 min) or monensin (100 µM, 40 mins). Samples probed for ATG2B and GFP. (F) Western blot analysis of GFP-IPs and input from RAW264.7 cells expressing GFP-LC3A following incubation with opsonized zymosan (OPZ) for 40 mins. Samples were probed for ATG2B and GFP. (G) Model of ATG8 response to lysosome damage illustrating the ATG8 associated lysosome tubulation and vesiculation, activation of the V-ATPase-ATG16L1 axis, ATG8 conjugation to PE and PS, and the engagement of lipid transfer protein ATG2. Image created using Biorender.

    Journal: bioRxiv

    Article Title: Lysosome damage triggers direct ATG8 conjugation and ATG2 engagement via CASM

    doi: 10.1101/2023.03.22.533754

    Figure Lengend Snippet: (A) Western blot analysis of GFP-IPs and input from wild type and ATG13 KO MCF10A cells expressing either GFP or GFP-LC3A treated +/- LLOMe (250 µM, 30 mins). Samples probed for ATG2B, GFP, ATG13 and GAPDH. (B) Western blot analysis of GFP-IPs and input from MCF10A ATG16L1 cell line panel expressing GFP-LC3A treated +/- LLOMe (250 µM, 30 mins). Samples probed for ATG2B, GFP, ATG16L1 and GAPDH. (C) Western blot analysis of GFP-IPs and total cell lysate (TCL) from GFP-LC3A expressing MCF10A cells treated with LLOMe (250 µM, 30 mins) or PP242 (1 µM, 1 hr) +/- Vps34 IN-1 (5 µM). Samples probed for ATG2B, GFP and GAPDH. (D) GFP-LC3A expressing ATG13 or ATG16L1 KO MCF10A cells were treated with LLOMe (250 µM, 30 mins). Following fractionation, input, cytosol and membrane fractions were probed for ATG2B, LAMP1, GFP and β-tubulin. (E) Western blot analysis of GFP-IPs and input from ATG13 KO MCF10A cells expressing GFP-LC3A treated with LLOMe (250 µM, 30mins), GPN (200 µM, 30 min) or monensin (100 µM, 40 mins). Samples probed for ATG2B and GFP. (F) Western blot analysis of GFP-IPs and input from RAW264.7 cells expressing GFP-LC3A following incubation with opsonized zymosan (OPZ) for 40 mins. Samples were probed for ATG2B and GFP. (G) Model of ATG8 response to lysosome damage illustrating the ATG8 associated lysosome tubulation and vesiculation, activation of the V-ATPase-ATG16L1 axis, ATG8 conjugation to PE and PS, and the engagement of lipid transfer protein ATG2. Image created using Biorender.

    Article Snippet: Antibodies used were rabbit polyclonal anti-ATG16L1 (8090, Cell Signalling, WB 1:1000), rabbit polyclonal anti-LC3A/B (4108, Cell Signalling, WB 1:1000, IF 1:100), rabbit monoclonal anti-ATP6V1A (ab199326, Abcam, WB 1:2000), mouse monoclonal anti-ATP6V0d1 (ab56441, Abcam, WB 1:1000), mouse monoclonal anti-LAMP1 (555798, BD Biosciences, IF 1:100), mouse monoclonal anti LAMP1 (611042, BD Bioscience, WB, 1:500), mouse monoclonal anti-GAPDH (ab8245, Abcam, WB 1:1000), rabbit anti-ATG13 (6940, Cell Signalling, WB 1:1000), mouse anti-GFP (1181446000, Roche, WB 1:1000), mouse anti-WIPI2 (MCA5780GA, BioRad, IF 1:100), rabbit anti ATG2B (25155-1-AP, ProteinTech, WB 1:1000), mouse anti-Galectin 3 (32790, Scbt, IF 1:100), rabbit anti-b tubulin (2128, Cell Signalling, WB 1:1000), Alexa Fluor 488 polyclonal goat anti-rabbit IgG (A-11034, ThermoFisher, IF 1:500), Alexa Fluor 568 polyclonal goat anti-mouse IgG (A-11004, ThermoFisher, IF 1:500), Alexa Fluor 568 polyclonal goat anti-rabbit IgG (A-11011, ThermoFisher, IF 1:500), HRP-conjugated anti-rabbit IgG (7074, Cell Signalling, WB 1:2000), HRP-conjugated anti-mouse IgG (7076, Cell Signalling, WB 1:2000).

    Techniques: Western Blot, Expressing, Fractionation, Incubation, Activation Assay, Conjugation Assay

    Nuclear egress phenotypes comparison of pUL13 K176A and pUS3 mutants (A-H) Shown are digital confocal images representing the localization of UL34 in infected Vero cells Green: pUL34. Cells were infected with HSV-1(F) (BAC), US3 K220A , UL13 K176A , and US3 mutants for 16 h at a MOI of 5, fixed at 16 hpi, and stained using antibodies directed against pUL34. Scale bar = 10μm. Representative images of one of three independent experiments are shown. (I) Frequencies of infected cell nuclei that show NEC aggregates. The error bars show the range of the values. Statistical significance was determined by one-way analysis of variance (ANOVA) using the Tukey method for multiple comparisons. ns, not significant; ***, P < 0.001. Data on graph represents at least three independent experiments. (J) US3 mobility shift assay of UL13 K176A and US3 mutants. Digital images of immunoblots for the indicated proteins in lysates of Vero cells infected for 18 h with 5 PFU/cell of BAC-derived WT HSV-1(F), US3 K220A or pUL13 K176A or US3 mutants. UL34 serves as a marker of equivalent infection and actin as a loading control.

    Journal: bioRxiv

    Article Title: DISTINCT ROLES OF VIRAL US3 AND UL13 PROTEIN KINASES IN HERPES VIRUS SIMPLEX TYPE 1 (HSV-1) NUCLEAR EGRESS

    doi: 10.1101/2023.03.20.533584

    Figure Lengend Snippet: Nuclear egress phenotypes comparison of pUL13 K176A and pUS3 mutants (A-H) Shown are digital confocal images representing the localization of UL34 in infected Vero cells Green: pUL34. Cells were infected with HSV-1(F) (BAC), US3 K220A , UL13 K176A , and US3 mutants for 16 h at a MOI of 5, fixed at 16 hpi, and stained using antibodies directed against pUL34. Scale bar = 10μm. Representative images of one of three independent experiments are shown. (I) Frequencies of infected cell nuclei that show NEC aggregates. The error bars show the range of the values. Statistical significance was determined by one-way analysis of variance (ANOVA) using the Tukey method for multiple comparisons. ns, not significant; ***, P < 0.001. Data on graph represents at least three independent experiments. (J) US3 mobility shift assay of UL13 K176A and US3 mutants. Digital images of immunoblots for the indicated proteins in lysates of Vero cells infected for 18 h with 5 PFU/cell of BAC-derived WT HSV-1(F), US3 K220A or pUL13 K176A or US3 mutants. UL34 serves as a marker of equivalent infection and actin as a loading control.

    Article Snippet: Cell lysates were separated with 10% SDS-PAGE gels, blotted onto nitrocellulose membranes, and probed for PRKAR2A using (mouse anti) rabbit anti-phospho-PKA substrates (1:500) (Cell Signaling), rabbit anti-phospho-Akt substrates (1:500) (Cell Signaling), mouse monoclonal anti-actin (Sigma-Aldrich), chicken polyclonal anti-UL34 (1:250) ( ).

    Techniques: Infection, Staining, Mobility Shift, Western Blot, Derivative Assay, Marker

    UL13 colocalizes with pUL31 inside the nucleus and interacts with pUL31 Shown are digital confocal images of representing the localization of FLAG-UL31, HA-UL34, and EGFP-UL13 transiently co-expressed proteins in Vero cells, single-cell transfection (A-C), without EGFP-UL13(D-G), or with EGFP-UL13 co-expression (H-K). Cells were fixed at 48 h post-transfection and were stained using antibodies directed against HA and FLAG. Green: EGFP-UL13, Blue: HA-UL34, Red: UL31-FLAG. Representative images of one of three independent experiments are shown. Bars, 5μm. (L) Co-immunoprecipitation of EGFP-UL13 and FLAG-UL31. 293-T cells were co-transfected with FLAG-UL31 and EGFP-UL13 plasmids. Cells were lysed after 48 hours post-transfection. Cell lysate samples were collected and incubated with FLAG magnetic beads to immunoprecipitate UL31 overnight. The whole lysate (WL) samples and immunoprecipitated (IP) eluents were separated by SDS-PAGE, blotted onto nitrocellulose membrane, and probed as indicated in the figure.

    Journal: bioRxiv

    Article Title: DISTINCT ROLES OF VIRAL US3 AND UL13 PROTEIN KINASES IN HERPES VIRUS SIMPLEX TYPE 1 (HSV-1) NUCLEAR EGRESS

    doi: 10.1101/2023.03.20.533584

    Figure Lengend Snippet: UL13 colocalizes with pUL31 inside the nucleus and interacts with pUL31 Shown are digital confocal images of representing the localization of FLAG-UL31, HA-UL34, and EGFP-UL13 transiently co-expressed proteins in Vero cells, single-cell transfection (A-C), without EGFP-UL13(D-G), or with EGFP-UL13 co-expression (H-K). Cells were fixed at 48 h post-transfection and were stained using antibodies directed against HA and FLAG. Green: EGFP-UL13, Blue: HA-UL34, Red: UL31-FLAG. Representative images of one of three independent experiments are shown. Bars, 5μm. (L) Co-immunoprecipitation of EGFP-UL13 and FLAG-UL31. 293-T cells were co-transfected with FLAG-UL31 and EGFP-UL13 plasmids. Cells were lysed after 48 hours post-transfection. Cell lysate samples were collected and incubated with FLAG magnetic beads to immunoprecipitate UL31 overnight. The whole lysate (WL) samples and immunoprecipitated (IP) eluents were separated by SDS-PAGE, blotted onto nitrocellulose membrane, and probed as indicated in the figure.

    Article Snippet: Cell lysates were separated with 10% SDS-PAGE gels, blotted onto nitrocellulose membranes, and probed for PRKAR2A using (mouse anti) rabbit anti-phospho-PKA substrates (1:500) (Cell Signaling), rabbit anti-phospho-Akt substrates (1:500) (Cell Signaling), mouse monoclonal anti-actin (Sigma-Aldrich), chicken polyclonal anti-UL34 (1:250) ( ).

    Techniques: Transfection, Expressing, Staining, Immunoprecipitation, Incubation, Magnetic Beads, SDS Page