polyclonal anti phospho cdc2 tyr15  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc polyclonal anti phospho cdc2 tyr15
    (A) Western blot analysis of 30 µg of cleared lysates from nocodazole-arrested HeLa cell which were treated with the indicated phosphatases for 1.5 h at 30°C. Phospho-Akt antibodies were used as a control of the enzymatic activity. (B) Cells arrested at mitosis with nocodazole were further treated with vehicle alone (−) or with the <t>Cdk1</t> inhibitor purvalanol A 10 mM (+) during 3 hours. Anti-lamin A/C antibodies were used to control loading. (C) Mitotic cells were treated with the MEK inhibitor PD98059 20 µM (+) or with vehicle alone (−) and cell lysates were analyzed by western blotting with AIB1 antibodies. Phospho-ERK specific antibodies were used as control for the efficiency of the treatment. (D) Western blot analysis of immunoprecipitated complexes using anti-AIB1 antibodies or pre-immune serum (Pre-I) from lysates of asynchronous HeLa cells (A) or from cells arrested with nocodazole (M). (E) Human AIB1 was subcloned in pFASTBac HTa and baculovirus were produced to express the recombinant full-length His6-AIB1 in Sf9 cells using the system BacPak (Clontech). Recombinant protein was purified by standard Ni 2+ -NTA affinity chromatography followed by desalting with a molecular exclusion column. Purified AIB1 (middle panel) was incubated with [γ- 32 P]ATP in the presence (+) or absence (−) of recombinant Cdk1/cyclin B1 for 1 hour at 30°C (upper panel). Reactions were resolved by SDS-PAGE, the gel was dryed and exposed to an X-ray film. 1 µg of histone H1 (Sigma) was used as a positive control (bottom panel).
    Polyclonal Anti Phospho Cdc2 Tyr15, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/polyclonal anti phospho cdc2 tyr15/product/Cell Signaling Technology Inc
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    polyclonal anti phospho cdc2 tyr15 - by Bioz Stars, 2023-02
    97/100 stars

    Images

    1) Product Images from "Phosphorylation of AIB1 at Mitosis Is Regulated by CDK1/CYCLIN B"

    Article Title: Phosphorylation of AIB1 at Mitosis Is Regulated by CDK1/CYCLIN B

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0028602

    (A) Western blot analysis of 30 µg of cleared lysates from nocodazole-arrested HeLa cell which were treated with the indicated phosphatases for 1.5 h at 30°C. Phospho-Akt antibodies were used as a control of the enzymatic activity. (B) Cells arrested at mitosis with nocodazole were further treated with vehicle alone (−) or with the Cdk1 inhibitor purvalanol A 10 mM (+) during 3 hours. Anti-lamin A/C antibodies were used to control loading. (C) Mitotic cells were treated with the MEK inhibitor PD98059 20 µM (+) or with vehicle alone (−) and cell lysates were analyzed by western blotting with AIB1 antibodies. Phospho-ERK specific antibodies were used as control for the efficiency of the treatment. (D) Western blot analysis of immunoprecipitated complexes using anti-AIB1 antibodies or pre-immune serum (Pre-I) from lysates of asynchronous HeLa cells (A) or from cells arrested with nocodazole (M). (E) Human AIB1 was subcloned in pFASTBac HTa and baculovirus were produced to express the recombinant full-length His6-AIB1 in Sf9 cells using the system BacPak (Clontech). Recombinant protein was purified by standard Ni 2+ -NTA affinity chromatography followed by desalting with a molecular exclusion column. Purified AIB1 (middle panel) was incubated with [γ- 32 P]ATP in the presence (+) or absence (−) of recombinant Cdk1/cyclin B1 for 1 hour at 30°C (upper panel). Reactions were resolved by SDS-PAGE, the gel was dryed and exposed to an X-ray film. 1 µg of histone H1 (Sigma) was used as a positive control (bottom panel).
    Figure Legend Snippet: (A) Western blot analysis of 30 µg of cleared lysates from nocodazole-arrested HeLa cell which were treated with the indicated phosphatases for 1.5 h at 30°C. Phospho-Akt antibodies were used as a control of the enzymatic activity. (B) Cells arrested at mitosis with nocodazole were further treated with vehicle alone (−) or with the Cdk1 inhibitor purvalanol A 10 mM (+) during 3 hours. Anti-lamin A/C antibodies were used to control loading. (C) Mitotic cells were treated with the MEK inhibitor PD98059 20 µM (+) or with vehicle alone (−) and cell lysates were analyzed by western blotting with AIB1 antibodies. Phospho-ERK specific antibodies were used as control for the efficiency of the treatment. (D) Western blot analysis of immunoprecipitated complexes using anti-AIB1 antibodies or pre-immune serum (Pre-I) from lysates of asynchronous HeLa cells (A) or from cells arrested with nocodazole (M). (E) Human AIB1 was subcloned in pFASTBac HTa and baculovirus were produced to express the recombinant full-length His6-AIB1 in Sf9 cells using the system BacPak (Clontech). Recombinant protein was purified by standard Ni 2+ -NTA affinity chromatography followed by desalting with a molecular exclusion column. Purified AIB1 (middle panel) was incubated with [γ- 32 P]ATP in the presence (+) or absence (−) of recombinant Cdk1/cyclin B1 for 1 hour at 30°C (upper panel). Reactions were resolved by SDS-PAGE, the gel was dryed and exposed to an X-ray film. 1 µg of histone H1 (Sigma) was used as a positive control (bottom panel).

    Techniques Used: Western Blot, Activity Assay, Immunoprecipitation, Produced, Recombinant, Purification, Affinity Chromatography, Incubation, SDS Page, Positive Control

    Floating HeLa cells arrested at mitosis with nocodazole were further treated for three more hours with vehicle alone as control (lane 1), 10 µM purvalanol A (lane 2), 1.25 µM Aurora kinase inhibitor II (lane 3), 7 µM Plk inhibitor III (lane 4) or 100 µM of the Hec1/Nek2 inhibitor I (lane 5). Cells were lysed and analyzed by westernblotting against the indicated molecular species. Importantly, exposure of cells to the Cdk1 inhibitor caused the desphosphorylation of AIB1 together with a rapid mitotic exit (lane 2) to a similar extent as previously reported with other inhibitors , . On the other hand, exposure of mitotic cells to inhibitors specific for Aurora A, B, Plks or Nek2 did not show any significant effect on AIB1 phosphorylation, suggesting that neither of them is a kinase for AIB1 under the experimental conditions tested. The protein kinase inhibitor staurosporine causes morphological changes at mitosis in HeLa cells, including the decondensation of chromosomes and the reformation of nuclear membrane . Treatment with the different kinase inhibitors at the concentrations indicated by the supplier caused similar morphological changes plus the reattachment of cells to the surface of the petri dish (data not shown), suggesting that the inhibitors indeed exerted a biologically relevant activity.
    Figure Legend Snippet: Floating HeLa cells arrested at mitosis with nocodazole were further treated for three more hours with vehicle alone as control (lane 1), 10 µM purvalanol A (lane 2), 1.25 µM Aurora kinase inhibitor II (lane 3), 7 µM Plk inhibitor III (lane 4) or 100 µM of the Hec1/Nek2 inhibitor I (lane 5). Cells were lysed and analyzed by westernblotting against the indicated molecular species. Importantly, exposure of cells to the Cdk1 inhibitor caused the desphosphorylation of AIB1 together with a rapid mitotic exit (lane 2) to a similar extent as previously reported with other inhibitors , . On the other hand, exposure of mitotic cells to inhibitors specific for Aurora A, B, Plks or Nek2 did not show any significant effect on AIB1 phosphorylation, suggesting that neither of them is a kinase for AIB1 under the experimental conditions tested. The protein kinase inhibitor staurosporine causes morphological changes at mitosis in HeLa cells, including the decondensation of chromosomes and the reformation of nuclear membrane . Treatment with the different kinase inhibitors at the concentrations indicated by the supplier caused similar morphological changes plus the reattachment of cells to the surface of the petri dish (data not shown), suggesting that the inhibitors indeed exerted a biologically relevant activity.

    Techniques Used: Activity Assay

    (A) Representative scheme of full length AIB1 and GST fusion fragments generated and named A through E. (B) Qualitative analysis of AIB1 fragments subjected to in vitro phosphorylation with active Cdk1/cyclin B. Reactions were resolved by SDS-PAGE and gel, dried on Whatman paper and exposed for autoradiography. Fragment containing amino acids 792–928 of the Retinoblastoma protein (Rb) was also expressed as a GST fussion protein and 1 µg was used in a parallel reaction as a positive control. (C) Same fragments as in (B) were also incubated with 100 ng of active Cdk1/Cyclin A2 (Cell Signaling). (D) Coomassie staining of the different AIB1 fragments fused to GST. Based in the cualitative amounts of proteins stained, double input of fragment E was used in the radioactive reactions. (E) Autoradiography (upper panel) of AIB1 fragment C (aminoacids 693–933) and fragments harbouring point substitutions serine 728 to alanine (S728A), serine 860 to alanine (S860A) and serine 867 to alanine (S867A), subjected to phosphorylation by complex Cdk1/cyclin B1 in the presence of [γ- 32 P]ATP, 1 hour at 30°C. Bottom panel represents a Coomassie staining of the fragments used in the upper kinase reactions. (F) Autoradiography exposure (upper panel) and coomassie staining (lower panel) of AIB1 fragment C and a mutant (C AYA) in which amino acids RYL localized at +11 from serine 728 are mutated to AYA. Fragments were incubated with active Cdk1/cyclin B1 for 1 hour at 30°C in the presence of [γ- 32 P]ATP. Reactions were resolved by SDS-PAGE and gel, dried on Whatman paper and exposed for autoradiography. Fragment containing amino acids 792–928 of the Retinoblastoma protein (Rb) was also expressed as a GST fusion protein and used in a parallel reaction as a positive control.
    Figure Legend Snippet: (A) Representative scheme of full length AIB1 and GST fusion fragments generated and named A through E. (B) Qualitative analysis of AIB1 fragments subjected to in vitro phosphorylation with active Cdk1/cyclin B. Reactions were resolved by SDS-PAGE and gel, dried on Whatman paper and exposed for autoradiography. Fragment containing amino acids 792–928 of the Retinoblastoma protein (Rb) was also expressed as a GST fussion protein and 1 µg was used in a parallel reaction as a positive control. (C) Same fragments as in (B) were also incubated with 100 ng of active Cdk1/Cyclin A2 (Cell Signaling). (D) Coomassie staining of the different AIB1 fragments fused to GST. Based in the cualitative amounts of proteins stained, double input of fragment E was used in the radioactive reactions. (E) Autoradiography (upper panel) of AIB1 fragment C (aminoacids 693–933) and fragments harbouring point substitutions serine 728 to alanine (S728A), serine 860 to alanine (S860A) and serine 867 to alanine (S867A), subjected to phosphorylation by complex Cdk1/cyclin B1 in the presence of [γ- 32 P]ATP, 1 hour at 30°C. Bottom panel represents a Coomassie staining of the fragments used in the upper kinase reactions. (F) Autoradiography exposure (upper panel) and coomassie staining (lower panel) of AIB1 fragment C and a mutant (C AYA) in which amino acids RYL localized at +11 from serine 728 are mutated to AYA. Fragments were incubated with active Cdk1/cyclin B1 for 1 hour at 30°C in the presence of [γ- 32 P]ATP. Reactions were resolved by SDS-PAGE and gel, dried on Whatman paper and exposed for autoradiography. Fragment containing amino acids 792–928 of the Retinoblastoma protein (Rb) was also expressed as a GST fusion protein and used in a parallel reaction as a positive control.

    Techniques Used: Generated, In Vitro, SDS Page, Autoradiography, Positive Control, Incubation, Staining, Mutagenesis

    polyclonal anti phospho cdc2 tyr15  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc polyclonal anti phospho cdc2 tyr15
    (A) Western blot analysis of 30 µg of cleared lysates from nocodazole-arrested HeLa cell which were treated with the indicated phosphatases for 1.5 h at 30°C. Phospho-Akt antibodies were used as a control of the enzymatic activity. (B) Cells arrested at mitosis with nocodazole were further treated with vehicle alone (−) or with the <t>Cdk1</t> inhibitor purvalanol A 10 mM (+) during 3 hours. Anti-lamin A/C antibodies were used to control loading. (C) Mitotic cells were treated with the MEK inhibitor PD98059 20 µM (+) or with vehicle alone (−) and cell lysates were analyzed by western blotting with AIB1 antibodies. Phospho-ERK specific antibodies were used as control for the efficiency of the treatment. (D) Western blot analysis of immunoprecipitated complexes using anti-AIB1 antibodies or pre-immune serum (Pre-I) from lysates of asynchronous HeLa cells (A) or from cells arrested with nocodazole (M). (E) Human AIB1 was subcloned in pFASTBac HTa and baculovirus were produced to express the recombinant full-length His6-AIB1 in Sf9 cells using the system BacPak (Clontech). Recombinant protein was purified by standard Ni 2+ -NTA affinity chromatography followed by desalting with a molecular exclusion column. Purified AIB1 (middle panel) was incubated with [γ- 32 P]ATP in the presence (+) or absence (−) of recombinant Cdk1/cyclin B1 for 1 hour at 30°C (upper panel). Reactions were resolved by SDS-PAGE, the gel was dryed and exposed to an X-ray film. 1 µg of histone H1 (Sigma) was used as a positive control (bottom panel).
    Polyclonal Anti Phospho Cdc2 Tyr15, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/polyclonal anti phospho cdc2 tyr15/product/Cell Signaling Technology Inc
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    polyclonal anti phospho cdc2 tyr15 - by Bioz Stars, 2023-02
    97/100 stars

    Images

    1) Product Images from "Phosphorylation of AIB1 at Mitosis Is Regulated by CDK1/CYCLIN B"

    Article Title: Phosphorylation of AIB1 at Mitosis Is Regulated by CDK1/CYCLIN B

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0028602

    (A) Western blot analysis of 30 µg of cleared lysates from nocodazole-arrested HeLa cell which were treated with the indicated phosphatases for 1.5 h at 30°C. Phospho-Akt antibodies were used as a control of the enzymatic activity. (B) Cells arrested at mitosis with nocodazole were further treated with vehicle alone (−) or with the Cdk1 inhibitor purvalanol A 10 mM (+) during 3 hours. Anti-lamin A/C antibodies were used to control loading. (C) Mitotic cells were treated with the MEK inhibitor PD98059 20 µM (+) or with vehicle alone (−) and cell lysates were analyzed by western blotting with AIB1 antibodies. Phospho-ERK specific antibodies were used as control for the efficiency of the treatment. (D) Western blot analysis of immunoprecipitated complexes using anti-AIB1 antibodies or pre-immune serum (Pre-I) from lysates of asynchronous HeLa cells (A) or from cells arrested with nocodazole (M). (E) Human AIB1 was subcloned in pFASTBac HTa and baculovirus were produced to express the recombinant full-length His6-AIB1 in Sf9 cells using the system BacPak (Clontech). Recombinant protein was purified by standard Ni 2+ -NTA affinity chromatography followed by desalting with a molecular exclusion column. Purified AIB1 (middle panel) was incubated with [γ- 32 P]ATP in the presence (+) or absence (−) of recombinant Cdk1/cyclin B1 for 1 hour at 30°C (upper panel). Reactions were resolved by SDS-PAGE, the gel was dryed and exposed to an X-ray film. 1 µg of histone H1 (Sigma) was used as a positive control (bottom panel).
    Figure Legend Snippet: (A) Western blot analysis of 30 µg of cleared lysates from nocodazole-arrested HeLa cell which were treated with the indicated phosphatases for 1.5 h at 30°C. Phospho-Akt antibodies were used as a control of the enzymatic activity. (B) Cells arrested at mitosis with nocodazole were further treated with vehicle alone (−) or with the Cdk1 inhibitor purvalanol A 10 mM (+) during 3 hours. Anti-lamin A/C antibodies were used to control loading. (C) Mitotic cells were treated with the MEK inhibitor PD98059 20 µM (+) or with vehicle alone (−) and cell lysates were analyzed by western blotting with AIB1 antibodies. Phospho-ERK specific antibodies were used as control for the efficiency of the treatment. (D) Western blot analysis of immunoprecipitated complexes using anti-AIB1 antibodies or pre-immune serum (Pre-I) from lysates of asynchronous HeLa cells (A) or from cells arrested with nocodazole (M). (E) Human AIB1 was subcloned in pFASTBac HTa and baculovirus were produced to express the recombinant full-length His6-AIB1 in Sf9 cells using the system BacPak (Clontech). Recombinant protein was purified by standard Ni 2+ -NTA affinity chromatography followed by desalting with a molecular exclusion column. Purified AIB1 (middle panel) was incubated with [γ- 32 P]ATP in the presence (+) or absence (−) of recombinant Cdk1/cyclin B1 for 1 hour at 30°C (upper panel). Reactions were resolved by SDS-PAGE, the gel was dryed and exposed to an X-ray film. 1 µg of histone H1 (Sigma) was used as a positive control (bottom panel).

    Techniques Used: Western Blot, Activity Assay, Immunoprecipitation, Produced, Recombinant, Purification, Affinity Chromatography, Incubation, SDS Page, Positive Control

    Floating HeLa cells arrested at mitosis with nocodazole were further treated for three more hours with vehicle alone as control (lane 1), 10 µM purvalanol A (lane 2), 1.25 µM Aurora kinase inhibitor II (lane 3), 7 µM Plk inhibitor III (lane 4) or 100 µM of the Hec1/Nek2 inhibitor I (lane 5). Cells were lysed and analyzed by westernblotting against the indicated molecular species. Importantly, exposure of cells to the Cdk1 inhibitor caused the desphosphorylation of AIB1 together with a rapid mitotic exit (lane 2) to a similar extent as previously reported with other inhibitors , . On the other hand, exposure of mitotic cells to inhibitors specific for Aurora A, B, Plks or Nek2 did not show any significant effect on AIB1 phosphorylation, suggesting that neither of them is a kinase for AIB1 under the experimental conditions tested. The protein kinase inhibitor staurosporine causes morphological changes at mitosis in HeLa cells, including the decondensation of chromosomes and the reformation of nuclear membrane . Treatment with the different kinase inhibitors at the concentrations indicated by the supplier caused similar morphological changes plus the reattachment of cells to the surface of the petri dish (data not shown), suggesting that the inhibitors indeed exerted a biologically relevant activity.
    Figure Legend Snippet: Floating HeLa cells arrested at mitosis with nocodazole were further treated for three more hours with vehicle alone as control (lane 1), 10 µM purvalanol A (lane 2), 1.25 µM Aurora kinase inhibitor II (lane 3), 7 µM Plk inhibitor III (lane 4) or 100 µM of the Hec1/Nek2 inhibitor I (lane 5). Cells were lysed and analyzed by westernblotting against the indicated molecular species. Importantly, exposure of cells to the Cdk1 inhibitor caused the desphosphorylation of AIB1 together with a rapid mitotic exit (lane 2) to a similar extent as previously reported with other inhibitors , . On the other hand, exposure of mitotic cells to inhibitors specific for Aurora A, B, Plks or Nek2 did not show any significant effect on AIB1 phosphorylation, suggesting that neither of them is a kinase for AIB1 under the experimental conditions tested. The protein kinase inhibitor staurosporine causes morphological changes at mitosis in HeLa cells, including the decondensation of chromosomes and the reformation of nuclear membrane . Treatment with the different kinase inhibitors at the concentrations indicated by the supplier caused similar morphological changes plus the reattachment of cells to the surface of the petri dish (data not shown), suggesting that the inhibitors indeed exerted a biologically relevant activity.

    Techniques Used: Activity Assay

    (A) Representative scheme of full length AIB1 and GST fusion fragments generated and named A through E. (B) Qualitative analysis of AIB1 fragments subjected to in vitro phosphorylation with active Cdk1/cyclin B. Reactions were resolved by SDS-PAGE and gel, dried on Whatman paper and exposed for autoradiography. Fragment containing amino acids 792–928 of the Retinoblastoma protein (Rb) was also expressed as a GST fussion protein and 1 µg was used in a parallel reaction as a positive control. (C) Same fragments as in (B) were also incubated with 100 ng of active Cdk1/Cyclin A2 (Cell Signaling). (D) Coomassie staining of the different AIB1 fragments fused to GST. Based in the cualitative amounts of proteins stained, double input of fragment E was used in the radioactive reactions. (E) Autoradiography (upper panel) of AIB1 fragment C (aminoacids 693–933) and fragments harbouring point substitutions serine 728 to alanine (S728A), serine 860 to alanine (S860A) and serine 867 to alanine (S867A), subjected to phosphorylation by complex Cdk1/cyclin B1 in the presence of [γ- 32 P]ATP, 1 hour at 30°C. Bottom panel represents a Coomassie staining of the fragments used in the upper kinase reactions. (F) Autoradiography exposure (upper panel) and coomassie staining (lower panel) of AIB1 fragment C and a mutant (C AYA) in which amino acids RYL localized at +11 from serine 728 are mutated to AYA. Fragments were incubated with active Cdk1/cyclin B1 for 1 hour at 30°C in the presence of [γ- 32 P]ATP. Reactions were resolved by SDS-PAGE and gel, dried on Whatman paper and exposed for autoradiography. Fragment containing amino acids 792–928 of the Retinoblastoma protein (Rb) was also expressed as a GST fusion protein and used in a parallel reaction as a positive control.
    Figure Legend Snippet: (A) Representative scheme of full length AIB1 and GST fusion fragments generated and named A through E. (B) Qualitative analysis of AIB1 fragments subjected to in vitro phosphorylation with active Cdk1/cyclin B. Reactions were resolved by SDS-PAGE and gel, dried on Whatman paper and exposed for autoradiography. Fragment containing amino acids 792–928 of the Retinoblastoma protein (Rb) was also expressed as a GST fussion protein and 1 µg was used in a parallel reaction as a positive control. (C) Same fragments as in (B) were also incubated with 100 ng of active Cdk1/Cyclin A2 (Cell Signaling). (D) Coomassie staining of the different AIB1 fragments fused to GST. Based in the cualitative amounts of proteins stained, double input of fragment E was used in the radioactive reactions. (E) Autoradiography (upper panel) of AIB1 fragment C (aminoacids 693–933) and fragments harbouring point substitutions serine 728 to alanine (S728A), serine 860 to alanine (S860A) and serine 867 to alanine (S867A), subjected to phosphorylation by complex Cdk1/cyclin B1 in the presence of [γ- 32 P]ATP, 1 hour at 30°C. Bottom panel represents a Coomassie staining of the fragments used in the upper kinase reactions. (F) Autoradiography exposure (upper panel) and coomassie staining (lower panel) of AIB1 fragment C and a mutant (C AYA) in which amino acids RYL localized at +11 from serine 728 are mutated to AYA. Fragments were incubated with active Cdk1/cyclin B1 for 1 hour at 30°C in the presence of [γ- 32 P]ATP. Reactions were resolved by SDS-PAGE and gel, dried on Whatman paper and exposed for autoradiography. Fragment containing amino acids 792–928 of the Retinoblastoma protein (Rb) was also expressed as a GST fusion protein and used in a parallel reaction as a positive control.

    Techniques Used: Generated, In Vitro, SDS Page, Autoradiography, Positive Control, Incubation, Staining, Mutagenesis

    pcdc2 tyr15  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc pcdc2 tyr15
    Pcdc2 Tyr15, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcdc2 tyr15/product/Cell Signaling Technology Inc
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pcdc2 tyr15 - by Bioz Stars, 2023-02
    97/100 stars

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    pi cdc2 tyr15  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc pi cdc2 tyr15
    ( A – B ) Immuno-staining of LNCaP and PC-3. Both cells were treated 75 nM LBH589 for 24 h. Immuno-staining ( A ) with <t>Cdc2</t> and Cdc25C antibodies and ( B ) of γ-tubulin (Green) and DAPI (Blue) in vehicle control (left) and LBH589 treatment (right) in LNCaP. ( C – D ) MEK inhibitor attenuated LBH589-induced prometaphase arrest in LNCaP. The cells were pre-treated 30 with UO126 for 30 minutes and sequentially treated with LBH589 for 24 h. ( C ) The cell cycles were analyzed by PI-staining and flow cytometry. ( D ) The metaphase cells were counted by DAPI staining presented as condensate chromatin.
    Pi Cdc2 Tyr15, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pi cdc2 tyr15/product/Cell Signaling Technology Inc
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pi cdc2 tyr15 - by Bioz Stars, 2023-02
    97/100 stars

    Images

    1) Product Images from "The HDAC Inhibitor LBH589 Induces ERK-Dependent Prometaphase Arrest in Prostate Cancer via HDAC6 Inactivation and Down-Regulation"

    Article Title: The HDAC Inhibitor LBH589 Induces ERK-Dependent Prometaphase Arrest in Prostate Cancer via HDAC6 Inactivation and Down-Regulation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0073401

    ( A – B ) Immuno-staining of LNCaP and PC-3. Both cells were treated 75 nM LBH589 for 24 h. Immuno-staining ( A ) with Cdc2 and Cdc25C antibodies and ( B ) of γ-tubulin (Green) and DAPI (Blue) in vehicle control (left) and LBH589 treatment (right) in LNCaP. ( C – D ) MEK inhibitor attenuated LBH589-induced prometaphase arrest in LNCaP. The cells were pre-treated 30 with UO126 for 30 minutes and sequentially treated with LBH589 for 24 h. ( C ) The cell cycles were analyzed by PI-staining and flow cytometry. ( D ) The metaphase cells were counted by DAPI staining presented as condensate chromatin.
    Figure Legend Snippet: ( A – B ) Immuno-staining of LNCaP and PC-3. Both cells were treated 75 nM LBH589 for 24 h. Immuno-staining ( A ) with Cdc2 and Cdc25C antibodies and ( B ) of γ-tubulin (Green) and DAPI (Blue) in vehicle control (left) and LBH589 treatment (right) in LNCaP. ( C – D ) MEK inhibitor attenuated LBH589-induced prometaphase arrest in LNCaP. The cells were pre-treated 30 with UO126 for 30 minutes and sequentially treated with LBH589 for 24 h. ( C ) The cell cycles were analyzed by PI-staining and flow cytometry. ( D ) The metaphase cells were counted by DAPI staining presented as condensate chromatin.

    Techniques Used: Immunostaining, Staining, Flow Cytometry

    phospho tyr15 cdc2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc phospho tyr15 cdc2
    Increased <t>Cdk1,</t> Cdc25C and CyclinB1 association with 14-3-3 θ but stable nucleocytoplasmic distribution during HIV- and Vpr-induced G 2 ,M arrest. Jurkat cells were infected as in Fig. 1 with RT- NL4-3 e-n-GFP virions either with (Vpr v ) or without (Δ) hVpr supplied in trans or with RT+ NL4-3 e-n-GFP (HIV; MOI 2). (A) Cell lysates were harvested two days post-infection for immunoprecipitation with 14-3-3 θ and immunoblotting for CyclinB1, Cdc25C-P.S216, Cdc25C, Cdk1-P.Y15, Cdk1, 14-3-3 θ, and Vpr as indicated. The 14-3-3 θ signal in the IP does not reflect poor immunoprecipitation of 14-3-3 θ but rather the result of membrane stripping prior to 14-3-3 θ blotting. (B) DNA content analysis (y-axis) is shown in flow cytometric dot plots against GFP (x-axis) on the right and as a histogram in the inset for the samples in (A). Note that the y-axis of the parent graph becomes the x-axis of the inset graph. The quadrant gate demarcates approximate G1 (lower) and S/G 2 ,M (upper) populations and the percentage of cells in each relevant quadrant is indicated. The DNA histogram profile analysis was separated into GFP-positive (+) and negative (-) populations by the x-axis gate for the HIV-infected culture; as expected the infected cells (+) show G 2 arrest, but the uninfected cells (-) are mostly G1. (C) G 2 ,M cell cycle arrest caused by Vpr v and HIV infection does not alter the cytoplasmic and nuclear distribution of 14-3-3 θ, Cdc25C, Cdk1, and CyclinB1. Jurkat T cells shown in (A-B) that were infected with NL4-3 e-n-GFP RT- Δ Vpr (Δ), RT- wt Vpr (Vpr v ), or NL4-3 e-n-GFP RT+ (HIV) for two days were lysed and biochemically separated into cytoplasmic and nuclear fractions. Lysate fractions were blotted as in (A) (the lower panel of Vpr blot represents a longer exposure in which Vpr v is more apparent), with the addition of probes for HIV-1 Vif, Poly(ADP-ribose) polymerase (PARP) as a nuclear marker, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a cytoplasmic loading control. Cell cycle profiles and GFP expression are shown in (B). (D) Viral lysates (20 μg) of RT- NL4-3 e-n-GFP virions with (+) or without (-) Vpr were western blotted for CyclinB1, Cdk1, p24, and Vpr as indicated.
    Phospho Tyr15 Cdc2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "14-3-3 theta binding to cell cycle regulatory factors is enhanced by HIV-1 Vpr"

    Article Title: 14-3-3 theta binding to cell cycle regulatory factors is enhanced by HIV-1 Vpr

    Journal: Biology Direct

    doi: 10.1186/1745-6150-3-17

    Increased Cdk1, Cdc25C and CyclinB1 association with 14-3-3 θ but stable nucleocytoplasmic distribution during HIV- and Vpr-induced G 2 ,M arrest. Jurkat cells were infected as in Fig. 1 with RT- NL4-3 e-n-GFP virions either with (Vpr v ) or without (Δ) hVpr supplied in trans or with RT+ NL4-3 e-n-GFP (HIV; MOI 2). (A) Cell lysates were harvested two days post-infection for immunoprecipitation with 14-3-3 θ and immunoblotting for CyclinB1, Cdc25C-P.S216, Cdc25C, Cdk1-P.Y15, Cdk1, 14-3-3 θ, and Vpr as indicated. The 14-3-3 θ signal in the IP does not reflect poor immunoprecipitation of 14-3-3 θ but rather the result of membrane stripping prior to 14-3-3 θ blotting. (B) DNA content analysis (y-axis) is shown in flow cytometric dot plots against GFP (x-axis) on the right and as a histogram in the inset for the samples in (A). Note that the y-axis of the parent graph becomes the x-axis of the inset graph. The quadrant gate demarcates approximate G1 (lower) and S/G 2 ,M (upper) populations and the percentage of cells in each relevant quadrant is indicated. The DNA histogram profile analysis was separated into GFP-positive (+) and negative (-) populations by the x-axis gate for the HIV-infected culture; as expected the infected cells (+) show G 2 arrest, but the uninfected cells (-) are mostly G1. (C) G 2 ,M cell cycle arrest caused by Vpr v and HIV infection does not alter the cytoplasmic and nuclear distribution of 14-3-3 θ, Cdc25C, Cdk1, and CyclinB1. Jurkat T cells shown in (A-B) that were infected with NL4-3 e-n-GFP RT- Δ Vpr (Δ), RT- wt Vpr (Vpr v ), or NL4-3 e-n-GFP RT+ (HIV) for two days were lysed and biochemically separated into cytoplasmic and nuclear fractions. Lysate fractions were blotted as in (A) (the lower panel of Vpr blot represents a longer exposure in which Vpr v is more apparent), with the addition of probes for HIV-1 Vif, Poly(ADP-ribose) polymerase (PARP) as a nuclear marker, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a cytoplasmic loading control. Cell cycle profiles and GFP expression are shown in (B). (D) Viral lysates (20 μg) of RT- NL4-3 e-n-GFP virions with (+) or without (-) Vpr were western blotted for CyclinB1, Cdk1, p24, and Vpr as indicated.
    Figure Legend Snippet: Increased Cdk1, Cdc25C and CyclinB1 association with 14-3-3 θ but stable nucleocytoplasmic distribution during HIV- and Vpr-induced G 2 ,M arrest. Jurkat cells were infected as in Fig. 1 with RT- NL4-3 e-n-GFP virions either with (Vpr v ) or without (Δ) hVpr supplied in trans or with RT+ NL4-3 e-n-GFP (HIV; MOI 2). (A) Cell lysates were harvested two days post-infection for immunoprecipitation with 14-3-3 θ and immunoblotting for CyclinB1, Cdc25C-P.S216, Cdc25C, Cdk1-P.Y15, Cdk1, 14-3-3 θ, and Vpr as indicated. The 14-3-3 θ signal in the IP does not reflect poor immunoprecipitation of 14-3-3 θ but rather the result of membrane stripping prior to 14-3-3 θ blotting. (B) DNA content analysis (y-axis) is shown in flow cytometric dot plots against GFP (x-axis) on the right and as a histogram in the inset for the samples in (A). Note that the y-axis of the parent graph becomes the x-axis of the inset graph. The quadrant gate demarcates approximate G1 (lower) and S/G 2 ,M (upper) populations and the percentage of cells in each relevant quadrant is indicated. The DNA histogram profile analysis was separated into GFP-positive (+) and negative (-) populations by the x-axis gate for the HIV-infected culture; as expected the infected cells (+) show G 2 arrest, but the uninfected cells (-) are mostly G1. (C) G 2 ,M cell cycle arrest caused by Vpr v and HIV infection does not alter the cytoplasmic and nuclear distribution of 14-3-3 θ, Cdc25C, Cdk1, and CyclinB1. Jurkat T cells shown in (A-B) that were infected with NL4-3 e-n-GFP RT- Δ Vpr (Δ), RT- wt Vpr (Vpr v ), or NL4-3 e-n-GFP RT+ (HIV) for two days were lysed and biochemically separated into cytoplasmic and nuclear fractions. Lysate fractions were blotted as in (A) (the lower panel of Vpr blot represents a longer exposure in which Vpr v is more apparent), with the addition of probes for HIV-1 Vif, Poly(ADP-ribose) polymerase (PARP) as a nuclear marker, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a cytoplasmic loading control. Cell cycle profiles and GFP expression are shown in (B). (D) Viral lysates (20 μg) of RT- NL4-3 e-n-GFP virions with (+) or without (-) Vpr were western blotted for CyclinB1, Cdk1, p24, and Vpr as indicated.

    Techniques Used: Infection, Immunoprecipitation, Western Blot, Stripping Membranes, Marker, Expressing

    Mutant Vpr fails to stimulate the association of CyclinB1, Cdk1, and Plk1 with 14-3-3 θ but still binds 14-3-3 θ itself. (A) Jurkat cells were infected as in Fig. 2 with RT- NL4-3 e-n-GFP virions (Vpr v ) lacking Vpr (Δ) or containing either wild-type (wt), R80A mutant (80A), or I70S mutant (70S) Vpr. Cells lysates were harvested two days post-infection. for immunoprecipitation with 14-3-3 θ antibody (IP: 14-3-3 θ) and the lysates (input) and IP were blotted as indicated with antibodies recognizing Plk1, CyclinB1, Cdk7, Cdk1, 14-3-3 θ, and Vpr. (B) DNA content analysis of the samples in (A), top row (Vpr v ), and of aphidicolin-synchronized Jurkat cells released from the G1 block for the indicated number of hours (bottom row; sync.). (C) Immunoprecipitation and western blot were performed as in (A) of lysates from the cell cycle synchronized cells shown in (B, bottom row). (D) Jurkat cells were infected with NL4-3 e-n-GFP (RT+) derivatives containing either wild-type Vpr and Vif (lane 1), Vpr but no Vif (lane 2), neither Vpr nor Vif (lane 3), or R80A mutant Vpr and no Vif (lane 4). Two days post-infection cells were lysed, immunoprecipitated, and immunoblotted as in (A) (top). Flow cytometric DNA content analysis was performed at the time of harvest and shown for the GFP+ (HIV-infected) population of each sample (bottom, numbering corresponds to lane numbers of blots). GFP expression is shown as an inset with the percentage of cells in the GFP-positive gate indicated within the plot.
    Figure Legend Snippet: Mutant Vpr fails to stimulate the association of CyclinB1, Cdk1, and Plk1 with 14-3-3 θ but still binds 14-3-3 θ itself. (A) Jurkat cells were infected as in Fig. 2 with RT- NL4-3 e-n-GFP virions (Vpr v ) lacking Vpr (Δ) or containing either wild-type (wt), R80A mutant (80A), or I70S mutant (70S) Vpr. Cells lysates were harvested two days post-infection. for immunoprecipitation with 14-3-3 θ antibody (IP: 14-3-3 θ) and the lysates (input) and IP were blotted as indicated with antibodies recognizing Plk1, CyclinB1, Cdk7, Cdk1, 14-3-3 θ, and Vpr. (B) DNA content analysis of the samples in (A), top row (Vpr v ), and of aphidicolin-synchronized Jurkat cells released from the G1 block for the indicated number of hours (bottom row; sync.). (C) Immunoprecipitation and western blot were performed as in (A) of lysates from the cell cycle synchronized cells shown in (B, bottom row). (D) Jurkat cells were infected with NL4-3 e-n-GFP (RT+) derivatives containing either wild-type Vpr and Vif (lane 1), Vpr but no Vif (lane 2), neither Vpr nor Vif (lane 3), or R80A mutant Vpr and no Vif (lane 4). Two days post-infection cells were lysed, immunoprecipitated, and immunoblotted as in (A) (top). Flow cytometric DNA content analysis was performed at the time of harvest and shown for the GFP+ (HIV-infected) population of each sample (bottom, numbering corresponds to lane numbers of blots). GFP expression is shown as an inset with the percentage of cells in the GFP-positive gate indicated within the plot.

    Techniques Used: Mutagenesis, Infection, Immunoprecipitation, Blocking Assay, Western Blot, Expressing

    Reduced 14-3-3 θ association with centrosomal proteins, centrin and Plk1, during HIV-1 infection-induced G 2 ,M arrest. (A) Jurkat cells were mock-infected (-) or infected with NL4-3 e-n-GFP derivatives encoding wild-type Vif and Vpr (wt), deleted Vpr (r-), deleted Vif (f-), or double deletion of Vpr and Vif (fr-) at an MOI of 1.5. Lysates were harvested two days post-infection and immunoprecipitated as in Fig. 2 (IP: 14-3-3 θ). Whole cell lysates (input) and IP samples were blotted for Plk1, 14-3-3 θ, centrin, Vif, and Vpr. There appeared to be poor transfer of centrin protein at the left edge of the gel (input lanes). The reduced band intensity for this sample does not reflect decreased centrin abundance as it was well-represented in the IP and similar experiments showed no changes in centrin expression upon HIV-1 infection. (B) DNA content analysis of the samples in (A) by propidium iodide staining. HIV-infected samples were pre-gated on GFP+ cells for DNA analysis. (C) Viability (large plot) and GFP expression by viable cells (inset) for samples in (A) and (B) were measured by flow cytometric detection of propidium iodide (PI) negative, large (high forward scatter) cells and GFP fluorescence (inset histogram), respectively, at the time lysates were harvested. Plots correspond to samples directly above in (B). The gates demarcate viable and GFP-positive cell populations and the percentage of cells within each gate is indicated. (D) Jurkat cells were mock-infected (m), infected with Vpr v as in (A), or infected with NL4-3 e-n-GFP (HIV) and harvested after 40 hours for immunoprecipitation with 14-3-3 θ and immunoblotting with importin β, CyclinB1, 14-3-3 θ, and Vpr. (E) DNA content analysis performed as in (B) is shown for the samples in (D). The inset histogram depicts the percentage of GFP+ cells expressing the NL4-3 e-n-GFP provirus. The DNA analysis for the HIV-infected sample was performed on the gated GFP population indicated. (F) Jurkat cells were mock-infected (m), infected with RT- NL4-3 e-n-GFP virions (Vpr v ) to deliver Vpr protein, or treated with adriamycin (adr). Cell lysates (input) were harvested after 40 hours for immunoprecipitation with importin β (IP) and immunoblotting with importin β, CyclinB1, Plk1, Cdk1, and Vpr as indicated. (G) Cell cycle analysis is shown for the samples in (F) at the time of lysis as measured by propidium iodide DNA staining as in (B).
    Figure Legend Snippet: Reduced 14-3-3 θ association with centrosomal proteins, centrin and Plk1, during HIV-1 infection-induced G 2 ,M arrest. (A) Jurkat cells were mock-infected (-) or infected with NL4-3 e-n-GFP derivatives encoding wild-type Vif and Vpr (wt), deleted Vpr (r-), deleted Vif (f-), or double deletion of Vpr and Vif (fr-) at an MOI of 1.5. Lysates were harvested two days post-infection and immunoprecipitated as in Fig. 2 (IP: 14-3-3 θ). Whole cell lysates (input) and IP samples were blotted for Plk1, 14-3-3 θ, centrin, Vif, and Vpr. There appeared to be poor transfer of centrin protein at the left edge of the gel (input lanes). The reduced band intensity for this sample does not reflect decreased centrin abundance as it was well-represented in the IP and similar experiments showed no changes in centrin expression upon HIV-1 infection. (B) DNA content analysis of the samples in (A) by propidium iodide staining. HIV-infected samples were pre-gated on GFP+ cells for DNA analysis. (C) Viability (large plot) and GFP expression by viable cells (inset) for samples in (A) and (B) were measured by flow cytometric detection of propidium iodide (PI) negative, large (high forward scatter) cells and GFP fluorescence (inset histogram), respectively, at the time lysates were harvested. Plots correspond to samples directly above in (B). The gates demarcate viable and GFP-positive cell populations and the percentage of cells within each gate is indicated. (D) Jurkat cells were mock-infected (m), infected with Vpr v as in (A), or infected with NL4-3 e-n-GFP (HIV) and harvested after 40 hours for immunoprecipitation with 14-3-3 θ and immunoblotting with importin β, CyclinB1, 14-3-3 θ, and Vpr. (E) DNA content analysis performed as in (B) is shown for the samples in (D). The inset histogram depicts the percentage of GFP+ cells expressing the NL4-3 e-n-GFP provirus. The DNA analysis for the HIV-infected sample was performed on the gated GFP population indicated. (F) Jurkat cells were mock-infected (m), infected with RT- NL4-3 e-n-GFP virions (Vpr v ) to deliver Vpr protein, or treated with adriamycin (adr). Cell lysates (input) were harvested after 40 hours for immunoprecipitation with importin β (IP) and immunoblotting with importin β, CyclinB1, Plk1, Cdk1, and Vpr as indicated. (G) Cell cycle analysis is shown for the samples in (F) at the time of lysis as measured by propidium iodide DNA staining as in (B).

    Techniques Used: Infection, Immunoprecipitation, Expressing, Staining, Fluorescence, Western Blot, Cell Cycle Assay, Lysis

    Cell cycle regulatory protein binding to 14-3-3 θ is also enhanced during G 2 ,M arrest induced by adriamycin. Jurkat T cells were treated with adriamycin (Adr; 0.2 μg/ml) for 24, 48, or 72 hours (h) or untreated (0) and examined for 14-3-3 θ co-immunoprecipitating (IP) proteins. (A) Western blot analysis of Plk1, CyclinB1, Cdk1, 14-3-3 θ and centrin in whole cell lysates before IP (input) and after 14-3-3 θ IP (IP: 14-3-3 θ) at the time of adriamycin treatment indicated. These data are representative of three independent experiments. Although the amount of 14-3-3 θ appears less in the untreated (0) time point of the IP, this was not a reproducible finding. (B) Viability of the samples used in the IP in (A) determined by flow cytometry detection of propidium iodide (PI) exclusion and large size (high forward scatter). The percentage of viable cells is indicated. Flow cytometric histograms of the DNA content for each sample determined by propidium iodide DNA staining (x-axis) are shown as an inset.
    Figure Legend Snippet: Cell cycle regulatory protein binding to 14-3-3 θ is also enhanced during G 2 ,M arrest induced by adriamycin. Jurkat T cells were treated with adriamycin (Adr; 0.2 μg/ml) for 24, 48, or 72 hours (h) or untreated (0) and examined for 14-3-3 θ co-immunoprecipitating (IP) proteins. (A) Western blot analysis of Plk1, CyclinB1, Cdk1, 14-3-3 θ and centrin in whole cell lysates before IP (input) and after 14-3-3 θ IP (IP: 14-3-3 θ) at the time of adriamycin treatment indicated. These data are representative of three independent experiments. Although the amount of 14-3-3 θ appears less in the untreated (0) time point of the IP, this was not a reproducible finding. (B) Viability of the samples used in the IP in (A) determined by flow cytometry detection of propidium iodide (PI) exclusion and large size (high forward scatter). The percentage of viable cells is indicated. Flow cytometric histograms of the DNA content for each sample determined by propidium iodide DNA staining (x-axis) are shown as an inset.

    Techniques Used: Protein Binding, Western Blot, Flow Cytometry, Staining

    Cell cycle regulatory proteins reside in the centrosome during G 2 ,M arrest induced by HIV-1 infection and adriamycin. (A) Western blot analysis of centrosomes isolated from Jurkat cells infected with NL4-3 e-n-GFP (HIV; MOI of 2) for two days. Centrosomes were isolated by discontinuous sucrose gradient and fractions were collected and separated by SDS-PAGE and western blotted for Plk1, CyclinB1, γ-tubulin, Cdk1, 14-3-3 θ, centrin, Vif, and Vpr. Lanes 1–6 represent fractions from the bottom of the gradient upward, with centrosomes most abundant in lane 3. Whole cell lysates were run in lane 8 and volume from the top of the gradient equivalent to that used for each fraction was run in lane 7 to demonstrate sedimentation of centrosomal proteins through the gradient.
    Figure Legend Snippet: Cell cycle regulatory proteins reside in the centrosome during G 2 ,M arrest induced by HIV-1 infection and adriamycin. (A) Western blot analysis of centrosomes isolated from Jurkat cells infected with NL4-3 e-n-GFP (HIV; MOI of 2) for two days. Centrosomes were isolated by discontinuous sucrose gradient and fractions were collected and separated by SDS-PAGE and western blotted for Plk1, CyclinB1, γ-tubulin, Cdk1, 14-3-3 θ, centrin, Vif, and Vpr. Lanes 1–6 represent fractions from the bottom of the gradient upward, with centrosomes most abundant in lane 3. Whole cell lysates were run in lane 8 and volume from the top of the gradient equivalent to that used for each fraction was run in lane 7 to demonstrate sedimentation of centrosomal proteins through the gradient. "L" indicates a light exposure and "D" indicates a darker exposure of the chemilumigraph of the middle part of the gel. (B) Viability (top) and DNA content analysis (bottom) by flow cytometry for the culture in (A; HIV (+)) and untreated Jurkats without (-) HIV infection. The percentage of viable cells was determined by propidium iodide (PI) exclusion and high forward scatter and is indicated in the lower right corner. Infection efficiency was measured by GFP expression (inset; gated population) and the percentage is indicated. DNA content was measured by flow cytometric detection of DNA stained with propidium iodide. The GFP-positive population was analyzed for the HIV-infected culture. (C) Jurkat T cells were treated with adriamycin (adr; 0.2 μg/ml; right panel) for two days or grown asynchronously (left panel) and centrosomes were isolated by discontinuous sucrose gradients as in (A). Centrosomes were most abundant in lanes 2–3 (untreated cells) or lanes 3–4 (adriamycin treated cells). Lanes were as described in panel A. (D) Cell cycle flow cytometric analysis of Jurkat cells in (C).

    Techniques Used: Infection, Western Blot, Isolation, SDS Page, Sedimentation, Flow Cytometry, Expressing, Staining

    anti cdc2 phospho tyr15  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti cdc2 phospho tyr15
    A, FACS analyses showed that the sub-G1 phase HuH7 cell population increased from approximately 0.7% to 7.7%, and cells in the G1 phase increased from approximately 69% to 87% 48 h after TGF-β1 treatment. Data represent the means ± S.D. of three experiments. * indicates significant differences between each group (P<0.05). B, <t>Cdc2</t> kinase activity was determined based on the level of phosphorylated histone H1 using histone H1 as a substrate. Cdc2 was activated 48 h after TGF-β1 treatment in HuH7 cells. However, cdc2 was not activated in HuH7R cells, which were isolated as an apoptosis-resistant clone from TGF-β1-treated HuH7 cells. Roscovitine (Ros)-pretreated HuH7 cells did not show cdc2 activation. A representative image from three independent experiments is shown. C, After TGF-β1 treatment (48 h), we observed cdc2 <t>Tyr15</t> dephosphorylation in association with Wee1 kinase down-regulation in apoptotic cells. Pretreatment with 20 µM roscovitine completely abolished apoptosis and restored Wee1 kinase expression. TGF-β1 treatment induced G1 cell cycle arrest in HuH7R cells; however, Wee1 kinase expression and non-phosphorylated cdc2 Tyr 15 were similar to those of roscovitine-pretreated HuH7 cells. A representative image of three experiments is shown. D, Wee1 down-regulation and cdc2 Tyr15 dephosphorylation commenced approximately 24 h after TGF-β1 treatment, which was similar to thea initiation of apoptosis. A representative Western blot image is shown in the upper panels. The results in the lower graphs represent the means ± S.D. of three experiments. * indicates significant differences between each group (P<0.05).
    Anti Cdc2 Phospho Tyr15, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Efficient Induction of Apoptosis by Wee1 Kinase Inhibition in Hepatocellular Carcinoma Cells"

    Article Title: Efficient Induction of Apoptosis by Wee1 Kinase Inhibition in Hepatocellular Carcinoma Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0100495

    A, FACS analyses showed that the sub-G1 phase HuH7 cell population increased from approximately 0.7% to 7.7%, and cells in the G1 phase increased from approximately 69% to 87% 48 h after TGF-β1 treatment. Data represent the means ± S.D. of three experiments. * indicates significant differences between each group (P<0.05). B, Cdc2 kinase activity was determined based on the level of phosphorylated histone H1 using histone H1 as a substrate. Cdc2 was activated 48 h after TGF-β1 treatment in HuH7 cells. However, cdc2 was not activated in HuH7R cells, which were isolated as an apoptosis-resistant clone from TGF-β1-treated HuH7 cells. Roscovitine (Ros)-pretreated HuH7 cells did not show cdc2 activation. A representative image from three independent experiments is shown. C, After TGF-β1 treatment (48 h), we observed cdc2 Tyr15 dephosphorylation in association with Wee1 kinase down-regulation in apoptotic cells. Pretreatment with 20 µM roscovitine completely abolished apoptosis and restored Wee1 kinase expression. TGF-β1 treatment induced G1 cell cycle arrest in HuH7R cells; however, Wee1 kinase expression and non-phosphorylated cdc2 Tyr 15 were similar to those of roscovitine-pretreated HuH7 cells. A representative image of three experiments is shown. D, Wee1 down-regulation and cdc2 Tyr15 dephosphorylation commenced approximately 24 h after TGF-β1 treatment, which was similar to thea initiation of apoptosis. A representative Western blot image is shown in the upper panels. The results in the lower graphs represent the means ± S.D. of three experiments. * indicates significant differences between each group (P<0.05).
    Figure Legend Snippet: A, FACS analyses showed that the sub-G1 phase HuH7 cell population increased from approximately 0.7% to 7.7%, and cells in the G1 phase increased from approximately 69% to 87% 48 h after TGF-β1 treatment. Data represent the means ± S.D. of three experiments. * indicates significant differences between each group (P<0.05). B, Cdc2 kinase activity was determined based on the level of phosphorylated histone H1 using histone H1 as a substrate. Cdc2 was activated 48 h after TGF-β1 treatment in HuH7 cells. However, cdc2 was not activated in HuH7R cells, which were isolated as an apoptosis-resistant clone from TGF-β1-treated HuH7 cells. Roscovitine (Ros)-pretreated HuH7 cells did not show cdc2 activation. A representative image from three independent experiments is shown. C, After TGF-β1 treatment (48 h), we observed cdc2 Tyr15 dephosphorylation in association with Wee1 kinase down-regulation in apoptotic cells. Pretreatment with 20 µM roscovitine completely abolished apoptosis and restored Wee1 kinase expression. TGF-β1 treatment induced G1 cell cycle arrest in HuH7R cells; however, Wee1 kinase expression and non-phosphorylated cdc2 Tyr 15 were similar to those of roscovitine-pretreated HuH7 cells. A representative image of three experiments is shown. D, Wee1 down-regulation and cdc2 Tyr15 dephosphorylation commenced approximately 24 h after TGF-β1 treatment, which was similar to thea initiation of apoptosis. A representative Western blot image is shown in the upper panels. The results in the lower graphs represent the means ± S.D. of three experiments. * indicates significant differences between each group (P<0.05).

    Techniques Used: Activity Assay, Isolation, Activation Assay, De-Phosphorylation Assay, Expressing, Western Blot

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    Phospho Cdk1 Tyr15, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    anti phospho cdc2 tyr15  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti phospho cdc2 tyr15
    Anti Phospho Cdc2 Tyr15, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    phospho cdc2 tyr15  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc phospho cdc2 tyr15
    (A) Flow cytometric determination of DNA content was analyzed using the Accuri C6 Flow Cytometer. shLASP-1-transfected HSC-3 and Ca9-22 cells show significant (* P <0.025; Mann-Whitney U test with Bonferroni correction) G2 phase accumulation as opposed to the shMock-transfected cells. (B) Immunoblotting analysis shows down-regulation of cyclin A and cyclin B and up-regulation <t>of</t> <t>phospho-cdc2</t> in the knockdown cells.
    Phospho Cdc2 Tyr15, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Overexpression of LIM and SH3 Protein 1 Leading to Accelerated G2/M Phase Transition Contributes to Enhanced Tumourigenesis in Oral Cancer"

    Article Title: Overexpression of LIM and SH3 Protein 1 Leading to Accelerated G2/M Phase Transition Contributes to Enhanced Tumourigenesis in Oral Cancer

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0083187

    (A) Flow cytometric determination of DNA content was analyzed using the Accuri C6 Flow Cytometer. shLASP-1-transfected HSC-3 and Ca9-22 cells show significant (* P <0.025; Mann-Whitney U test with Bonferroni correction) G2 phase accumulation as opposed to the shMock-transfected cells. (B) Immunoblotting analysis shows down-regulation of cyclin A and cyclin B and up-regulation of phospho-cdc2 in the knockdown cells.
    Figure Legend Snippet: (A) Flow cytometric determination of DNA content was analyzed using the Accuri C6 Flow Cytometer. shLASP-1-transfected HSC-3 and Ca9-22 cells show significant (* P <0.025; Mann-Whitney U test with Bonferroni correction) G2 phase accumulation as opposed to the shMock-transfected cells. (B) Immunoblotting analysis shows down-regulation of cyclin A and cyclin B and up-regulation of phospho-cdc2 in the knockdown cells.

    Techniques Used: Flow Cytometry, Transfection, MANN-WHITNEY, Western Blot

    phospho cdk1 tyr15  (Cell Signaling Technology Inc)


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    Structured Review

    Cell Signaling Technology Inc phospho cdk1 tyr15
    Inhibition of WEE1 by the small molecule inhibitor MK-1775. (A) A decrease in relative cell number was seen by MTS assay through a wide range of MK-1775 concentrations in both Daoy and UW228 cells. From this data the IC50 values were calculated. (B) Treatment with an IC30 of MK-1775 for 48 hours significantly decreased the relative colony numbers in Daoy and UW228 cells. Shown are representative images from each treatment group with the quantifying bar graph for each cell line below the images. (C) Exposure to increasing concentrations of MK-1775 in Daoy and UW228 cells showed a dose dependent decrease in WEE1 activity as seen by decreased <t>phospho-CDK1</t> <t>(Tyr15).</t> There was no significant change in WEE1 levels. (D) Treatment with MK-1775 is sufficient to decrease subcutaneous tumor growth of Daoy cells in mice. Mice were treated on three consecutive days a week for the first three weeks after tumor establishment.
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    1) Product Images from "Integrated genomic analysis identifies the mitotic checkpoint kinase WEE1 as a novel therapeutic target in medulloblastoma"

    Article Title: Integrated genomic analysis identifies the mitotic checkpoint kinase WEE1 as a novel therapeutic target in medulloblastoma

    Journal: Molecular Cancer

    doi: 10.1186/1476-4598-13-72

    Inhibition of WEE1 by the small molecule inhibitor MK-1775. (A) A decrease in relative cell number was seen by MTS assay through a wide range of MK-1775 concentrations in both Daoy and UW228 cells. From this data the IC50 values were calculated. (B) Treatment with an IC30 of MK-1775 for 48 hours significantly decreased the relative colony numbers in Daoy and UW228 cells. Shown are representative images from each treatment group with the quantifying bar graph for each cell line below the images. (C) Exposure to increasing concentrations of MK-1775 in Daoy and UW228 cells showed a dose dependent decrease in WEE1 activity as seen by decreased phospho-CDK1 (Tyr15). There was no significant change in WEE1 levels. (D) Treatment with MK-1775 is sufficient to decrease subcutaneous tumor growth of Daoy cells in mice. Mice were treated on three consecutive days a week for the first three weeks after tumor establishment.
    Figure Legend Snippet: Inhibition of WEE1 by the small molecule inhibitor MK-1775. (A) A decrease in relative cell number was seen by MTS assay through a wide range of MK-1775 concentrations in both Daoy and UW228 cells. From this data the IC50 values were calculated. (B) Treatment with an IC30 of MK-1775 for 48 hours significantly decreased the relative colony numbers in Daoy and UW228 cells. Shown are representative images from each treatment group with the quantifying bar graph for each cell line below the images. (C) Exposure to increasing concentrations of MK-1775 in Daoy and UW228 cells showed a dose dependent decrease in WEE1 activity as seen by decreased phospho-CDK1 (Tyr15). There was no significant change in WEE1 levels. (D) Treatment with MK-1775 is sufficient to decrease subcutaneous tumor growth of Daoy cells in mice. Mice were treated on three consecutive days a week for the first three weeks after tumor establishment.

    Techniques Used: Inhibition, MTS Assay, Activity Assay

    anti phospho cdc2 tyr15  (Cell Signaling Technology Inc)


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    Structured Review

    Cell Signaling Technology Inc anti phospho cdc2 tyr15
    H1229 cells treated with empty vector + IC 20 dose of cisplatin (3.0 µM), NPRL2, or NPRL2+ IC 20 dose of cisplatin were harvested at 24, 48, and 72 h after treatment and analyzed by Western blotting for expression of cell cycle signaling molecules. Control H1299 cells were treated with empty vector and harvested at 72 h after treatment. β-actin was used as a loading control. ( A ) Cdc25A was degraded by the treatment of NPRL2 or IC 20 dose of cisplatin 72 h later, and this degradation in treatment of NPRL2+ cisplatin strongly appeared 24 to 72 h later. P-Cdc25C was slightly increased by treatment with cisplatin; in contrast, it was remarkably increased by treatment with NPRL2 or NPRL2+ cisplatin. <t>P-Cdc2</t> and P-SMC1 were clearly enhanced more with NPRL2+ cisplatin treatment than with cisplatin or NPRL2 treatment. ( B ) Immunoprecipitation Western blotting (IP-WB) analysis for protein-protein interaction between Cdc2-cyclin B1. H1299 cells were transfected with either empty vector or NPRL2 plasmid with or without IC 20 value of cisplatin. The backbone plasmid vector without NPRL2 was used as a transfection control. Protein extracts were collected 72 h after transfection and immunoprecipitated with either anti-Cdc2 or anti-cyclin B1 antibody and immunoblotted with Cdc2 or cyclin B1 antibody. NPRL2 and cisplatin treatment remarkably degraded the interaction of Cdc2/cyclin B1 complex.
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    1) Product Images from "NPRL2 Sensitizes Human Non-Small Cell Lung Cancer (NSCLC) Cells to Cisplatin Treatment by Regulating Key Components in the DNA Repair Pathway"

    Article Title: NPRL2 Sensitizes Human Non-Small Cell Lung Cancer (NSCLC) Cells to Cisplatin Treatment by Regulating Key Components in the DNA Repair Pathway

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0011994

    H1229 cells treated with empty vector + IC 20 dose of cisplatin (3.0 µM), NPRL2, or NPRL2+ IC 20 dose of cisplatin were harvested at 24, 48, and 72 h after treatment and analyzed by Western blotting for expression of cell cycle signaling molecules. Control H1299 cells were treated with empty vector and harvested at 72 h after treatment. β-actin was used as a loading control. ( A ) Cdc25A was degraded by the treatment of NPRL2 or IC 20 dose of cisplatin 72 h later, and this degradation in treatment of NPRL2+ cisplatin strongly appeared 24 to 72 h later. P-Cdc25C was slightly increased by treatment with cisplatin; in contrast, it was remarkably increased by treatment with NPRL2 or NPRL2+ cisplatin. P-Cdc2 and P-SMC1 were clearly enhanced more with NPRL2+ cisplatin treatment than with cisplatin or NPRL2 treatment. ( B ) Immunoprecipitation Western blotting (IP-WB) analysis for protein-protein interaction between Cdc2-cyclin B1. H1299 cells were transfected with either empty vector or NPRL2 plasmid with or without IC 20 value of cisplatin. The backbone plasmid vector without NPRL2 was used as a transfection control. Protein extracts were collected 72 h after transfection and immunoprecipitated with either anti-Cdc2 or anti-cyclin B1 antibody and immunoblotted with Cdc2 or cyclin B1 antibody. NPRL2 and cisplatin treatment remarkably degraded the interaction of Cdc2/cyclin B1 complex.
    Figure Legend Snippet: H1229 cells treated with empty vector + IC 20 dose of cisplatin (3.0 µM), NPRL2, or NPRL2+ IC 20 dose of cisplatin were harvested at 24, 48, and 72 h after treatment and analyzed by Western blotting for expression of cell cycle signaling molecules. Control H1299 cells were treated with empty vector and harvested at 72 h after treatment. β-actin was used as a loading control. ( A ) Cdc25A was degraded by the treatment of NPRL2 or IC 20 dose of cisplatin 72 h later, and this degradation in treatment of NPRL2+ cisplatin strongly appeared 24 to 72 h later. P-Cdc25C was slightly increased by treatment with cisplatin; in contrast, it was remarkably increased by treatment with NPRL2 or NPRL2+ cisplatin. P-Cdc2 and P-SMC1 were clearly enhanced more with NPRL2+ cisplatin treatment than with cisplatin or NPRL2 treatment. ( B ) Immunoprecipitation Western blotting (IP-WB) analysis for protein-protein interaction between Cdc2-cyclin B1. H1299 cells were transfected with either empty vector or NPRL2 plasmid with or without IC 20 value of cisplatin. The backbone plasmid vector without NPRL2 was used as a transfection control. Protein extracts were collected 72 h after transfection and immunoprecipitated with either anti-Cdc2 or anti-cyclin B1 antibody and immunoblotted with Cdc2 or cyclin B1 antibody. NPRL2 and cisplatin treatment remarkably degraded the interaction of Cdc2/cyclin B1 complex.

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

    NPRL2+ cisplatin ( cis -diamminedichloroplatinum (II) [CDDP]) treatment effector caspase, caspase-3, is activated and PARP is cleaved. Therefore, our result that the combination treatment of NPRL2 and CDDP activates a caspase cascade and hyperphosphorylates H2AX suggests that this combination treatment can strongly enhance the apoptotic pathway. Adding NPRL2 treatment to CDDP significantly enhanced Chk2 and Chk1 kinase activity, and NPRL2+ CDDP treatment remarkably degraded the interaction of Cdc2 and cyclin B1, leading to the inactivation of the Cdc2/cyclin B1 complex and arrest in G2/M. The cells arrested at the G2/M phase ultimately promote apoptosis with characteristic nuclear fragmentation. This combination treatment strongly inactivates Cdc25A and Cdc25C, and phospho-Cdc2 (Tyr-15), an inactivated type of Cdc2, is clearly increased in NPRL2+ CDDP treatment.
    Figure Legend Snippet: NPRL2+ cisplatin ( cis -diamminedichloroplatinum (II) [CDDP]) treatment effector caspase, caspase-3, is activated and PARP is cleaved. Therefore, our result that the combination treatment of NPRL2 and CDDP activates a caspase cascade and hyperphosphorylates H2AX suggests that this combination treatment can strongly enhance the apoptotic pathway. Adding NPRL2 treatment to CDDP significantly enhanced Chk2 and Chk1 kinase activity, and NPRL2+ CDDP treatment remarkably degraded the interaction of Cdc2 and cyclin B1, leading to the inactivation of the Cdc2/cyclin B1 complex and arrest in G2/M. The cells arrested at the G2/M phase ultimately promote apoptosis with characteristic nuclear fragmentation. This combination treatment strongly inactivates Cdc25A and Cdc25C, and phospho-Cdc2 (Tyr-15), an inactivated type of Cdc2, is clearly increased in NPRL2+ CDDP treatment.

    Techniques Used: Activity Assay

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    Cell Signaling Technology Inc polyclonal anti phospho cdc2 tyr15
    (A) Western blot analysis of 30 µg of cleared lysates from nocodazole-arrested HeLa cell which were treated with the indicated phosphatases for 1.5 h at 30°C. Phospho-Akt antibodies were used as a control of the enzymatic activity. (B) Cells arrested at mitosis with nocodazole were further treated with vehicle alone (−) or with the <t>Cdk1</t> inhibitor purvalanol A 10 mM (+) during 3 hours. Anti-lamin A/C antibodies were used to control loading. (C) Mitotic cells were treated with the MEK inhibitor PD98059 20 µM (+) or with vehicle alone (−) and cell lysates were analyzed by western blotting with AIB1 antibodies. Phospho-ERK specific antibodies were used as control for the efficiency of the treatment. (D) Western blot analysis of immunoprecipitated complexes using anti-AIB1 antibodies or pre-immune serum (Pre-I) from lysates of asynchronous HeLa cells (A) or from cells arrested with nocodazole (M). (E) Human AIB1 was subcloned in pFASTBac HTa and baculovirus were produced to express the recombinant full-length His6-AIB1 in Sf9 cells using the system BacPak (Clontech). Recombinant protein was purified by standard Ni 2+ -NTA affinity chromatography followed by desalting with a molecular exclusion column. Purified AIB1 (middle panel) was incubated with [γ- 32 P]ATP in the presence (+) or absence (−) of recombinant Cdk1/cyclin B1 for 1 hour at 30°C (upper panel). Reactions were resolved by SDS-PAGE, the gel was dryed and exposed to an X-ray film. 1 µg of histone H1 (Sigma) was used as a positive control (bottom panel).
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    Cell Signaling Technology Inc pcdc2 tyr15
    (A) Western blot analysis of 30 µg of cleared lysates from nocodazole-arrested HeLa cell which were treated with the indicated phosphatases for 1.5 h at 30°C. Phospho-Akt antibodies were used as a control of the enzymatic activity. (B) Cells arrested at mitosis with nocodazole were further treated with vehicle alone (−) or with the <t>Cdk1</t> inhibitor purvalanol A 10 mM (+) during 3 hours. Anti-lamin A/C antibodies were used to control loading. (C) Mitotic cells were treated with the MEK inhibitor PD98059 20 µM (+) or with vehicle alone (−) and cell lysates were analyzed by western blotting with AIB1 antibodies. Phospho-ERK specific antibodies were used as control for the efficiency of the treatment. (D) Western blot analysis of immunoprecipitated complexes using anti-AIB1 antibodies or pre-immune serum (Pre-I) from lysates of asynchronous HeLa cells (A) or from cells arrested with nocodazole (M). (E) Human AIB1 was subcloned in pFASTBac HTa and baculovirus were produced to express the recombinant full-length His6-AIB1 in Sf9 cells using the system BacPak (Clontech). Recombinant protein was purified by standard Ni 2+ -NTA affinity chromatography followed by desalting with a molecular exclusion column. Purified AIB1 (middle panel) was incubated with [γ- 32 P]ATP in the presence (+) or absence (−) of recombinant Cdk1/cyclin B1 for 1 hour at 30°C (upper panel). Reactions were resolved by SDS-PAGE, the gel was dryed and exposed to an X-ray film. 1 µg of histone H1 (Sigma) was used as a positive control (bottom panel).
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    Cell Signaling Technology Inc pi cdc2 tyr15
    ( A – B ) Immuno-staining of LNCaP and PC-3. Both cells were treated 75 nM LBH589 for 24 h. Immuno-staining ( A ) with <t>Cdc2</t> and Cdc25C antibodies and ( B ) of γ-tubulin (Green) and DAPI (Blue) in vehicle control (left) and LBH589 treatment (right) in LNCaP. ( C – D ) MEK inhibitor attenuated LBH589-induced prometaphase arrest in LNCaP. The cells were pre-treated 30 with UO126 for 30 minutes and sequentially treated with LBH589 for 24 h. ( C ) The cell cycles were analyzed by PI-staining and flow cytometry. ( D ) The metaphase cells were counted by DAPI staining presented as condensate chromatin.
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    Cell Signaling Technology Inc phospho tyr15 cdc2
    Increased <t>Cdk1,</t> Cdc25C and CyclinB1 association with 14-3-3 θ but stable nucleocytoplasmic distribution during HIV- and Vpr-induced G 2 ,M arrest. Jurkat cells were infected as in Fig. 1 with RT- NL4-3 e-n-GFP virions either with (Vpr v ) or without (Δ) hVpr supplied in trans or with RT+ NL4-3 e-n-GFP (HIV; MOI 2). (A) Cell lysates were harvested two days post-infection for immunoprecipitation with 14-3-3 θ and immunoblotting for CyclinB1, Cdc25C-P.S216, Cdc25C, Cdk1-P.Y15, Cdk1, 14-3-3 θ, and Vpr as indicated. The 14-3-3 θ signal in the IP does not reflect poor immunoprecipitation of 14-3-3 θ but rather the result of membrane stripping prior to 14-3-3 θ blotting. (B) DNA content analysis (y-axis) is shown in flow cytometric dot plots against GFP (x-axis) on the right and as a histogram in the inset for the samples in (A). Note that the y-axis of the parent graph becomes the x-axis of the inset graph. The quadrant gate demarcates approximate G1 (lower) and S/G 2 ,M (upper) populations and the percentage of cells in each relevant quadrant is indicated. The DNA histogram profile analysis was separated into GFP-positive (+) and negative (-) populations by the x-axis gate for the HIV-infected culture; as expected the infected cells (+) show G 2 arrest, but the uninfected cells (-) are mostly G1. (C) G 2 ,M cell cycle arrest caused by Vpr v and HIV infection does not alter the cytoplasmic and nuclear distribution of 14-3-3 θ, Cdc25C, Cdk1, and CyclinB1. Jurkat T cells shown in (A-B) that were infected with NL4-3 e-n-GFP RT- Δ Vpr (Δ), RT- wt Vpr (Vpr v ), or NL4-3 e-n-GFP RT+ (HIV) for two days were lysed and biochemically separated into cytoplasmic and nuclear fractions. Lysate fractions were blotted as in (A) (the lower panel of Vpr blot represents a longer exposure in which Vpr v is more apparent), with the addition of probes for HIV-1 Vif, Poly(ADP-ribose) polymerase (PARP) as a nuclear marker, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a cytoplasmic loading control. Cell cycle profiles and GFP expression are shown in (B). (D) Viral lysates (20 μg) of RT- NL4-3 e-n-GFP virions with (+) or without (-) Vpr were western blotted for CyclinB1, Cdk1, p24, and Vpr as indicated.
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    Cell Signaling Technology Inc anti cdc2 phospho tyr15
    A, FACS analyses showed that the sub-G1 phase HuH7 cell population increased from approximately 0.7% to 7.7%, and cells in the G1 phase increased from approximately 69% to 87% 48 h after TGF-β1 treatment. Data represent the means ± S.D. of three experiments. * indicates significant differences between each group (P<0.05). B, <t>Cdc2</t> kinase activity was determined based on the level of phosphorylated histone H1 using histone H1 as a substrate. Cdc2 was activated 48 h after TGF-β1 treatment in HuH7 cells. However, cdc2 was not activated in HuH7R cells, which were isolated as an apoptosis-resistant clone from TGF-β1-treated HuH7 cells. Roscovitine (Ros)-pretreated HuH7 cells did not show cdc2 activation. A representative image from three independent experiments is shown. C, After TGF-β1 treatment (48 h), we observed cdc2 <t>Tyr15</t> dephosphorylation in association with Wee1 kinase down-regulation in apoptotic cells. Pretreatment with 20 µM roscovitine completely abolished apoptosis and restored Wee1 kinase expression. TGF-β1 treatment induced G1 cell cycle arrest in HuH7R cells; however, Wee1 kinase expression and non-phosphorylated cdc2 Tyr 15 were similar to those of roscovitine-pretreated HuH7 cells. A representative image of three experiments is shown. D, Wee1 down-regulation and cdc2 Tyr15 dephosphorylation commenced approximately 24 h after TGF-β1 treatment, which was similar to thea initiation of apoptosis. A representative Western blot image is shown in the upper panels. The results in the lower graphs represent the means ± S.D. of three experiments. * indicates significant differences between each group (P<0.05).
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    Cell Signaling Technology Inc phospho cdk1 tyr15
    A, FACS analyses showed that the sub-G1 phase HuH7 cell population increased from approximately 0.7% to 7.7%, and cells in the G1 phase increased from approximately 69% to 87% 48 h after TGF-β1 treatment. Data represent the means ± S.D. of three experiments. * indicates significant differences between each group (P<0.05). B, <t>Cdc2</t> kinase activity was determined based on the level of phosphorylated histone H1 using histone H1 as a substrate. Cdc2 was activated 48 h after TGF-β1 treatment in HuH7 cells. However, cdc2 was not activated in HuH7R cells, which were isolated as an apoptosis-resistant clone from TGF-β1-treated HuH7 cells. Roscovitine (Ros)-pretreated HuH7 cells did not show cdc2 activation. A representative image from three independent experiments is shown. C, After TGF-β1 treatment (48 h), we observed cdc2 <t>Tyr15</t> dephosphorylation in association with Wee1 kinase down-regulation in apoptotic cells. Pretreatment with 20 µM roscovitine completely abolished apoptosis and restored Wee1 kinase expression. TGF-β1 treatment induced G1 cell cycle arrest in HuH7R cells; however, Wee1 kinase expression and non-phosphorylated cdc2 Tyr 15 were similar to those of roscovitine-pretreated HuH7 cells. A representative image of three experiments is shown. D, Wee1 down-regulation and cdc2 Tyr15 dephosphorylation commenced approximately 24 h after TGF-β1 treatment, which was similar to thea initiation of apoptosis. A representative Western blot image is shown in the upper panels. The results in the lower graphs represent the means ± S.D. of three experiments. * indicates significant differences between each group (P<0.05).
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    Cell Signaling Technology Inc anti phospho cdc2 tyr15
    A, FACS analyses showed that the sub-G1 phase HuH7 cell population increased from approximately 0.7% to 7.7%, and cells in the G1 phase increased from approximately 69% to 87% 48 h after TGF-β1 treatment. Data represent the means ± S.D. of three experiments. * indicates significant differences between each group (P<0.05). B, <t>Cdc2</t> kinase activity was determined based on the level of phosphorylated histone H1 using histone H1 as a substrate. Cdc2 was activated 48 h after TGF-β1 treatment in HuH7 cells. However, cdc2 was not activated in HuH7R cells, which were isolated as an apoptosis-resistant clone from TGF-β1-treated HuH7 cells. Roscovitine (Ros)-pretreated HuH7 cells did not show cdc2 activation. A representative image from three independent experiments is shown. C, After TGF-β1 treatment (48 h), we observed cdc2 <t>Tyr15</t> dephosphorylation in association with Wee1 kinase down-regulation in apoptotic cells. Pretreatment with 20 µM roscovitine completely abolished apoptosis and restored Wee1 kinase expression. TGF-β1 treatment induced G1 cell cycle arrest in HuH7R cells; however, Wee1 kinase expression and non-phosphorylated cdc2 Tyr 15 were similar to those of roscovitine-pretreated HuH7 cells. A representative image of three experiments is shown. D, Wee1 down-regulation and cdc2 Tyr15 dephosphorylation commenced approximately 24 h after TGF-β1 treatment, which was similar to thea initiation of apoptosis. A representative Western blot image is shown in the upper panels. The results in the lower graphs represent the means ± S.D. of three experiments. * indicates significant differences between each group (P<0.05).
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    Cell Signaling Technology Inc phospho cdc2 tyr15
    (A) Flow cytometric determination of DNA content was analyzed using the Accuri C6 Flow Cytometer. shLASP-1-transfected HSC-3 and Ca9-22 cells show significant (* P <0.025; Mann-Whitney U test with Bonferroni correction) G2 phase accumulation as opposed to the shMock-transfected cells. (B) Immunoblotting analysis shows down-regulation of cyclin A and cyclin B and up-regulation <t>of</t> <t>phospho-cdc2</t> in the knockdown cells.
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    (A) Western blot analysis of 30 µg of cleared lysates from nocodazole-arrested HeLa cell which were treated with the indicated phosphatases for 1.5 h at 30°C. Phospho-Akt antibodies were used as a control of the enzymatic activity. (B) Cells arrested at mitosis with nocodazole were further treated with vehicle alone (−) or with the Cdk1 inhibitor purvalanol A 10 mM (+) during 3 hours. Anti-lamin A/C antibodies were used to control loading. (C) Mitotic cells were treated with the MEK inhibitor PD98059 20 µM (+) or with vehicle alone (−) and cell lysates were analyzed by western blotting with AIB1 antibodies. Phospho-ERK specific antibodies were used as control for the efficiency of the treatment. (D) Western blot analysis of immunoprecipitated complexes using anti-AIB1 antibodies or pre-immune serum (Pre-I) from lysates of asynchronous HeLa cells (A) or from cells arrested with nocodazole (M). (E) Human AIB1 was subcloned in pFASTBac HTa and baculovirus were produced to express the recombinant full-length His6-AIB1 in Sf9 cells using the system BacPak (Clontech). Recombinant protein was purified by standard Ni 2+ -NTA affinity chromatography followed by desalting with a molecular exclusion column. Purified AIB1 (middle panel) was incubated with [γ- 32 P]ATP in the presence (+) or absence (−) of recombinant Cdk1/cyclin B1 for 1 hour at 30°C (upper panel). Reactions were resolved by SDS-PAGE, the gel was dryed and exposed to an X-ray film. 1 µg of histone H1 (Sigma) was used as a positive control (bottom panel).

    Journal: PLoS ONE

    Article Title: Phosphorylation of AIB1 at Mitosis Is Regulated by CDK1/CYCLIN B

    doi: 10.1371/journal.pone.0028602

    Figure Lengend Snippet: (A) Western blot analysis of 30 µg of cleared lysates from nocodazole-arrested HeLa cell which were treated with the indicated phosphatases for 1.5 h at 30°C. Phospho-Akt antibodies were used as a control of the enzymatic activity. (B) Cells arrested at mitosis with nocodazole were further treated with vehicle alone (−) or with the Cdk1 inhibitor purvalanol A 10 mM (+) during 3 hours. Anti-lamin A/C antibodies were used to control loading. (C) Mitotic cells were treated with the MEK inhibitor PD98059 20 µM (+) or with vehicle alone (−) and cell lysates were analyzed by western blotting with AIB1 antibodies. Phospho-ERK specific antibodies were used as control for the efficiency of the treatment. (D) Western blot analysis of immunoprecipitated complexes using anti-AIB1 antibodies or pre-immune serum (Pre-I) from lysates of asynchronous HeLa cells (A) or from cells arrested with nocodazole (M). (E) Human AIB1 was subcloned in pFASTBac HTa and baculovirus were produced to express the recombinant full-length His6-AIB1 in Sf9 cells using the system BacPak (Clontech). Recombinant protein was purified by standard Ni 2+ -NTA affinity chromatography followed by desalting with a molecular exclusion column. Purified AIB1 (middle panel) was incubated with [γ- 32 P]ATP in the presence (+) or absence (−) of recombinant Cdk1/cyclin B1 for 1 hour at 30°C (upper panel). Reactions were resolved by SDS-PAGE, the gel was dryed and exposed to an X-ray film. 1 µg of histone H1 (Sigma) was used as a positive control (bottom panel).

    Article Snippet: Samples were resolved in 7.5% or 15% SDS-PAGE, blotted to Immobilon-P membranes (Millipore) and probed with the following antibodies: monoclonal anti-AIB1 (BD Biosciences), polyclonal anti-phospho-AKT Ser473, polyclonal anti-phospho-Cdc2 Tyr15, polyclonal anti-phospho-PP1 Thr320 and polyclonal anti-Cdc2 (Cell Signaling); monoclonal anti-phospho-ERK1/2 Tyr204, polyclonal anti-cyclin A, polyclonal anti-cyclin B1, monoclonal anti-cyclin D1, polyclonal anti-cyclin E, polyclonal anti-PP1, monoclonal anti-Lamin A/C from (Santa Cruz Biotech); monoclonal anti-phospho-histone H3 from Millipore; monoclonal anti-β-actin from Sigma and monoclonal anti-ubiquitin FK2 (Affinity Bioreagents).

    Techniques: Western Blot, Activity Assay, Immunoprecipitation, Produced, Recombinant, Purification, Affinity Chromatography, Incubation, SDS Page, Positive Control

    Floating HeLa cells arrested at mitosis with nocodazole were further treated for three more hours with vehicle alone as control (lane 1), 10 µM purvalanol A (lane 2), 1.25 µM Aurora kinase inhibitor II (lane 3), 7 µM Plk inhibitor III (lane 4) or 100 µM of the Hec1/Nek2 inhibitor I (lane 5). Cells were lysed and analyzed by westernblotting against the indicated molecular species. Importantly, exposure of cells to the Cdk1 inhibitor caused the desphosphorylation of AIB1 together with a rapid mitotic exit (lane 2) to a similar extent as previously reported with other inhibitors , . On the other hand, exposure of mitotic cells to inhibitors specific for Aurora A, B, Plks or Nek2 did not show any significant effect on AIB1 phosphorylation, suggesting that neither of them is a kinase for AIB1 under the experimental conditions tested. The protein kinase inhibitor staurosporine causes morphological changes at mitosis in HeLa cells, including the decondensation of chromosomes and the reformation of nuclear membrane . Treatment with the different kinase inhibitors at the concentrations indicated by the supplier caused similar morphological changes plus the reattachment of cells to the surface of the petri dish (data not shown), suggesting that the inhibitors indeed exerted a biologically relevant activity.

    Journal: PLoS ONE

    Article Title: Phosphorylation of AIB1 at Mitosis Is Regulated by CDK1/CYCLIN B

    doi: 10.1371/journal.pone.0028602

    Figure Lengend Snippet: Floating HeLa cells arrested at mitosis with nocodazole were further treated for three more hours with vehicle alone as control (lane 1), 10 µM purvalanol A (lane 2), 1.25 µM Aurora kinase inhibitor II (lane 3), 7 µM Plk inhibitor III (lane 4) or 100 µM of the Hec1/Nek2 inhibitor I (lane 5). Cells were lysed and analyzed by westernblotting against the indicated molecular species. Importantly, exposure of cells to the Cdk1 inhibitor caused the desphosphorylation of AIB1 together with a rapid mitotic exit (lane 2) to a similar extent as previously reported with other inhibitors , . On the other hand, exposure of mitotic cells to inhibitors specific for Aurora A, B, Plks or Nek2 did not show any significant effect on AIB1 phosphorylation, suggesting that neither of them is a kinase for AIB1 under the experimental conditions tested. The protein kinase inhibitor staurosporine causes morphological changes at mitosis in HeLa cells, including the decondensation of chromosomes and the reformation of nuclear membrane . Treatment with the different kinase inhibitors at the concentrations indicated by the supplier caused similar morphological changes plus the reattachment of cells to the surface of the petri dish (data not shown), suggesting that the inhibitors indeed exerted a biologically relevant activity.

    Article Snippet: Samples were resolved in 7.5% or 15% SDS-PAGE, blotted to Immobilon-P membranes (Millipore) and probed with the following antibodies: monoclonal anti-AIB1 (BD Biosciences), polyclonal anti-phospho-AKT Ser473, polyclonal anti-phospho-Cdc2 Tyr15, polyclonal anti-phospho-PP1 Thr320 and polyclonal anti-Cdc2 (Cell Signaling); monoclonal anti-phospho-ERK1/2 Tyr204, polyclonal anti-cyclin A, polyclonal anti-cyclin B1, monoclonal anti-cyclin D1, polyclonal anti-cyclin E, polyclonal anti-PP1, monoclonal anti-Lamin A/C from (Santa Cruz Biotech); monoclonal anti-phospho-histone H3 from Millipore; monoclonal anti-β-actin from Sigma and monoclonal anti-ubiquitin FK2 (Affinity Bioreagents).

    Techniques: Activity Assay

    (A) Representative scheme of full length AIB1 and GST fusion fragments generated and named A through E. (B) Qualitative analysis of AIB1 fragments subjected to in vitro phosphorylation with active Cdk1/cyclin B. Reactions were resolved by SDS-PAGE and gel, dried on Whatman paper and exposed for autoradiography. Fragment containing amino acids 792–928 of the Retinoblastoma protein (Rb) was also expressed as a GST fussion protein and 1 µg was used in a parallel reaction as a positive control. (C) Same fragments as in (B) were also incubated with 100 ng of active Cdk1/Cyclin A2 (Cell Signaling). (D) Coomassie staining of the different AIB1 fragments fused to GST. Based in the cualitative amounts of proteins stained, double input of fragment E was used in the radioactive reactions. (E) Autoradiography (upper panel) of AIB1 fragment C (aminoacids 693–933) and fragments harbouring point substitutions serine 728 to alanine (S728A), serine 860 to alanine (S860A) and serine 867 to alanine (S867A), subjected to phosphorylation by complex Cdk1/cyclin B1 in the presence of [γ- 32 P]ATP, 1 hour at 30°C. Bottom panel represents a Coomassie staining of the fragments used in the upper kinase reactions. (F) Autoradiography exposure (upper panel) and coomassie staining (lower panel) of AIB1 fragment C and a mutant (C AYA) in which amino acids RYL localized at +11 from serine 728 are mutated to AYA. Fragments were incubated with active Cdk1/cyclin B1 for 1 hour at 30°C in the presence of [γ- 32 P]ATP. Reactions were resolved by SDS-PAGE and gel, dried on Whatman paper and exposed for autoradiography. Fragment containing amino acids 792–928 of the Retinoblastoma protein (Rb) was also expressed as a GST fusion protein and used in a parallel reaction as a positive control.

    Journal: PLoS ONE

    Article Title: Phosphorylation of AIB1 at Mitosis Is Regulated by CDK1/CYCLIN B

    doi: 10.1371/journal.pone.0028602

    Figure Lengend Snippet: (A) Representative scheme of full length AIB1 and GST fusion fragments generated and named A through E. (B) Qualitative analysis of AIB1 fragments subjected to in vitro phosphorylation with active Cdk1/cyclin B. Reactions were resolved by SDS-PAGE and gel, dried on Whatman paper and exposed for autoradiography. Fragment containing amino acids 792–928 of the Retinoblastoma protein (Rb) was also expressed as a GST fussion protein and 1 µg was used in a parallel reaction as a positive control. (C) Same fragments as in (B) were also incubated with 100 ng of active Cdk1/Cyclin A2 (Cell Signaling). (D) Coomassie staining of the different AIB1 fragments fused to GST. Based in the cualitative amounts of proteins stained, double input of fragment E was used in the radioactive reactions. (E) Autoradiography (upper panel) of AIB1 fragment C (aminoacids 693–933) and fragments harbouring point substitutions serine 728 to alanine (S728A), serine 860 to alanine (S860A) and serine 867 to alanine (S867A), subjected to phosphorylation by complex Cdk1/cyclin B1 in the presence of [γ- 32 P]ATP, 1 hour at 30°C. Bottom panel represents a Coomassie staining of the fragments used in the upper kinase reactions. (F) Autoradiography exposure (upper panel) and coomassie staining (lower panel) of AIB1 fragment C and a mutant (C AYA) in which amino acids RYL localized at +11 from serine 728 are mutated to AYA. Fragments were incubated with active Cdk1/cyclin B1 for 1 hour at 30°C in the presence of [γ- 32 P]ATP. Reactions were resolved by SDS-PAGE and gel, dried on Whatman paper and exposed for autoradiography. Fragment containing amino acids 792–928 of the Retinoblastoma protein (Rb) was also expressed as a GST fusion protein and used in a parallel reaction as a positive control.

    Article Snippet: Samples were resolved in 7.5% or 15% SDS-PAGE, blotted to Immobilon-P membranes (Millipore) and probed with the following antibodies: monoclonal anti-AIB1 (BD Biosciences), polyclonal anti-phospho-AKT Ser473, polyclonal anti-phospho-Cdc2 Tyr15, polyclonal anti-phospho-PP1 Thr320 and polyclonal anti-Cdc2 (Cell Signaling); monoclonal anti-phospho-ERK1/2 Tyr204, polyclonal anti-cyclin A, polyclonal anti-cyclin B1, monoclonal anti-cyclin D1, polyclonal anti-cyclin E, polyclonal anti-PP1, monoclonal anti-Lamin A/C from (Santa Cruz Biotech); monoclonal anti-phospho-histone H3 from Millipore; monoclonal anti-β-actin from Sigma and monoclonal anti-ubiquitin FK2 (Affinity Bioreagents).

    Techniques: Generated, In Vitro, SDS Page, Autoradiography, Positive Control, Incubation, Staining, Mutagenesis

    ( A – B ) Immuno-staining of LNCaP and PC-3. Both cells were treated 75 nM LBH589 for 24 h. Immuno-staining ( A ) with Cdc2 and Cdc25C antibodies and ( B ) of γ-tubulin (Green) and DAPI (Blue) in vehicle control (left) and LBH589 treatment (right) in LNCaP. ( C – D ) MEK inhibitor attenuated LBH589-induced prometaphase arrest in LNCaP. The cells were pre-treated 30 with UO126 for 30 minutes and sequentially treated with LBH589 for 24 h. ( C ) The cell cycles were analyzed by PI-staining and flow cytometry. ( D ) The metaphase cells were counted by DAPI staining presented as condensate chromatin.

    Journal: PLoS ONE

    Article Title: The HDAC Inhibitor LBH589 Induces ERK-Dependent Prometaphase Arrest in Prostate Cancer via HDAC6 Inactivation and Down-Regulation

    doi: 10.1371/journal.pone.0073401

    Figure Lengend Snippet: ( A – B ) Immuno-staining of LNCaP and PC-3. Both cells were treated 75 nM LBH589 for 24 h. Immuno-staining ( A ) with Cdc2 and Cdc25C antibodies and ( B ) of γ-tubulin (Green) and DAPI (Blue) in vehicle control (left) and LBH589 treatment (right) in LNCaP. ( C – D ) MEK inhibitor attenuated LBH589-induced prometaphase arrest in LNCaP. The cells were pre-treated 30 with UO126 for 30 minutes and sequentially treated with LBH589 for 24 h. ( C ) The cell cycles were analyzed by PI-staining and flow cytometry. ( D ) The metaphase cells were counted by DAPI staining presented as condensate chromatin.

    Article Snippet: The antibodies included HDAC1 (#2062), c-Raf (#9422), Akt (#9272), Pi-Akt (#4058), Pi-Cdc25C (Ser216) (#9528), Pi-c-Raf (S259) (# 9421), Pi-c-Raf (S338) (#9427), and Pi-Cdc2 (Tyr15) (#9111) purchased from Cell Signaling Technology (Beverly, MA, USA); HDAC6 (sc-28386), Pi-ERK(sc-7383), ERK (sc-94), 14-3-3ζ (sc-1019), and Cdc2 (sc-54) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); Aurora A (#07-648), H3-Ac (#06-599), H4-Ac (#06-598), p21 (#05-345), and PP2A (#05-421) from Millipore (Lake Placid, NY, USA); GAPDH and actin (Sigma-Aldrich); Aurora B (#1788-1), Cdc25C (#1302-1), and PP1 (#1950-1) from Epitomics (Burlingame, CA); and HDAC3 (ab32369) from Abcam (Cambridge, UK).

    Techniques: Immunostaining, Staining, Flow Cytometry

    Increased Cdk1, Cdc25C and CyclinB1 association with 14-3-3 θ but stable nucleocytoplasmic distribution during HIV- and Vpr-induced G 2 ,M arrest. Jurkat cells were infected as in Fig. 1 with RT- NL4-3 e-n-GFP virions either with (Vpr v ) or without (Δ) hVpr supplied in trans or with RT+ NL4-3 e-n-GFP (HIV; MOI 2). (A) Cell lysates were harvested two days post-infection for immunoprecipitation with 14-3-3 θ and immunoblotting for CyclinB1, Cdc25C-P.S216, Cdc25C, Cdk1-P.Y15, Cdk1, 14-3-3 θ, and Vpr as indicated. The 14-3-3 θ signal in the IP does not reflect poor immunoprecipitation of 14-3-3 θ but rather the result of membrane stripping prior to 14-3-3 θ blotting. (B) DNA content analysis (y-axis) is shown in flow cytometric dot plots against GFP (x-axis) on the right and as a histogram in the inset for the samples in (A). Note that the y-axis of the parent graph becomes the x-axis of the inset graph. The quadrant gate demarcates approximate G1 (lower) and S/G 2 ,M (upper) populations and the percentage of cells in each relevant quadrant is indicated. The DNA histogram profile analysis was separated into GFP-positive (+) and negative (-) populations by the x-axis gate for the HIV-infected culture; as expected the infected cells (+) show G 2 arrest, but the uninfected cells (-) are mostly G1. (C) G 2 ,M cell cycle arrest caused by Vpr v and HIV infection does not alter the cytoplasmic and nuclear distribution of 14-3-3 θ, Cdc25C, Cdk1, and CyclinB1. Jurkat T cells shown in (A-B) that were infected with NL4-3 e-n-GFP RT- Δ Vpr (Δ), RT- wt Vpr (Vpr v ), or NL4-3 e-n-GFP RT+ (HIV) for two days were lysed and biochemically separated into cytoplasmic and nuclear fractions. Lysate fractions were blotted as in (A) (the lower panel of Vpr blot represents a longer exposure in which Vpr v is more apparent), with the addition of probes for HIV-1 Vif, Poly(ADP-ribose) polymerase (PARP) as a nuclear marker, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a cytoplasmic loading control. Cell cycle profiles and GFP expression are shown in (B). (D) Viral lysates (20 μg) of RT- NL4-3 e-n-GFP virions with (+) or without (-) Vpr were western blotted for CyclinB1, Cdk1, p24, and Vpr as indicated.

    Journal: Biology Direct

    Article Title: 14-3-3 theta binding to cell cycle regulatory factors is enhanced by HIV-1 Vpr

    doi: 10.1186/1745-6150-3-17

    Figure Lengend Snippet: Increased Cdk1, Cdc25C and CyclinB1 association with 14-3-3 θ but stable nucleocytoplasmic distribution during HIV- and Vpr-induced G 2 ,M arrest. Jurkat cells were infected as in Fig. 1 with RT- NL4-3 e-n-GFP virions either with (Vpr v ) or without (Δ) hVpr supplied in trans or with RT+ NL4-3 e-n-GFP (HIV; MOI 2). (A) Cell lysates were harvested two days post-infection for immunoprecipitation with 14-3-3 θ and immunoblotting for CyclinB1, Cdc25C-P.S216, Cdc25C, Cdk1-P.Y15, Cdk1, 14-3-3 θ, and Vpr as indicated. The 14-3-3 θ signal in the IP does not reflect poor immunoprecipitation of 14-3-3 θ but rather the result of membrane stripping prior to 14-3-3 θ blotting. (B) DNA content analysis (y-axis) is shown in flow cytometric dot plots against GFP (x-axis) on the right and as a histogram in the inset for the samples in (A). Note that the y-axis of the parent graph becomes the x-axis of the inset graph. The quadrant gate demarcates approximate G1 (lower) and S/G 2 ,M (upper) populations and the percentage of cells in each relevant quadrant is indicated. The DNA histogram profile analysis was separated into GFP-positive (+) and negative (-) populations by the x-axis gate for the HIV-infected culture; as expected the infected cells (+) show G 2 arrest, but the uninfected cells (-) are mostly G1. (C) G 2 ,M cell cycle arrest caused by Vpr v and HIV infection does not alter the cytoplasmic and nuclear distribution of 14-3-3 θ, Cdc25C, Cdk1, and CyclinB1. Jurkat T cells shown in (A-B) that were infected with NL4-3 e-n-GFP RT- Δ Vpr (Δ), RT- wt Vpr (Vpr v ), or NL4-3 e-n-GFP RT+ (HIV) for two days were lysed and biochemically separated into cytoplasmic and nuclear fractions. Lysate fractions were blotted as in (A) (the lower panel of Vpr blot represents a longer exposure in which Vpr v is more apparent), with the addition of probes for HIV-1 Vif, Poly(ADP-ribose) polymerase (PARP) as a nuclear marker, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a cytoplasmic loading control. Cell cycle profiles and GFP expression are shown in (B). (D) Viral lysates (20 μg) of RT- NL4-3 e-n-GFP virions with (+) or without (-) Vpr were western blotted for CyclinB1, Cdk1, p24, and Vpr as indicated.

    Article Snippet: Antibodies used with corresponding catalog numbers are as follows: β-actin (Sigma; A5441), Cdc2 (Santa Cruz; sc-54), Phospho-Tyr15-Cdc2 (Cell Signaling; 9111), Cdc25C (BD Pharmingen; 550922), Cdc25C (Santa Cruz; sc-327), Cdc25C (Calbiochem; CC26; EMSA), Phospho-Ser216-Cdc25C (Cell Signaling; 4901), Centrin (Sigma; C7736), CyclinB1 (Santa Cruz; sc-752 or sc-245), GAPDH (Abcam; ab9484), PARP (BD Transduction Laboratories; P76420), importin β (Sigma; I2534) PKA (Santa Cruz; sc-903), Plk1 (Upstate; 05–844), γ-Tubulin (Sigma; T3559); HIV-1 Vif (NIH ARRRP; 6459), HIV-1 Vpr (gift of K. Strebel, NIAID), 14-3-3 β (Santa Cruz; sc-25276), 14-3-3 θ (Santa Cruz; sc-732), 14-3-3 γ (Santa Cruz; sc-731c), 14-3-3 ζ (Santa Cruz; sc-1019; cross-reacts with β and σ isoforms).

    Techniques: Infection, Immunoprecipitation, Western Blot, Stripping Membranes, Marker, Expressing

    Mutant Vpr fails to stimulate the association of CyclinB1, Cdk1, and Plk1 with 14-3-3 θ but still binds 14-3-3 θ itself. (A) Jurkat cells were infected as in Fig. 2 with RT- NL4-3 e-n-GFP virions (Vpr v ) lacking Vpr (Δ) or containing either wild-type (wt), R80A mutant (80A), or I70S mutant (70S) Vpr. Cells lysates were harvested two days post-infection. for immunoprecipitation with 14-3-3 θ antibody (IP: 14-3-3 θ) and the lysates (input) and IP were blotted as indicated with antibodies recognizing Plk1, CyclinB1, Cdk7, Cdk1, 14-3-3 θ, and Vpr. (B) DNA content analysis of the samples in (A), top row (Vpr v ), and of aphidicolin-synchronized Jurkat cells released from the G1 block for the indicated number of hours (bottom row; sync.). (C) Immunoprecipitation and western blot were performed as in (A) of lysates from the cell cycle synchronized cells shown in (B, bottom row). (D) Jurkat cells were infected with NL4-3 e-n-GFP (RT+) derivatives containing either wild-type Vpr and Vif (lane 1), Vpr but no Vif (lane 2), neither Vpr nor Vif (lane 3), or R80A mutant Vpr and no Vif (lane 4). Two days post-infection cells were lysed, immunoprecipitated, and immunoblotted as in (A) (top). Flow cytometric DNA content analysis was performed at the time of harvest and shown for the GFP+ (HIV-infected) population of each sample (bottom, numbering corresponds to lane numbers of blots). GFP expression is shown as an inset with the percentage of cells in the GFP-positive gate indicated within the plot.

    Journal: Biology Direct

    Article Title: 14-3-3 theta binding to cell cycle regulatory factors is enhanced by HIV-1 Vpr

    doi: 10.1186/1745-6150-3-17

    Figure Lengend Snippet: Mutant Vpr fails to stimulate the association of CyclinB1, Cdk1, and Plk1 with 14-3-3 θ but still binds 14-3-3 θ itself. (A) Jurkat cells were infected as in Fig. 2 with RT- NL4-3 e-n-GFP virions (Vpr v ) lacking Vpr (Δ) or containing either wild-type (wt), R80A mutant (80A), or I70S mutant (70S) Vpr. Cells lysates were harvested two days post-infection. for immunoprecipitation with 14-3-3 θ antibody (IP: 14-3-3 θ) and the lysates (input) and IP were blotted as indicated with antibodies recognizing Plk1, CyclinB1, Cdk7, Cdk1, 14-3-3 θ, and Vpr. (B) DNA content analysis of the samples in (A), top row (Vpr v ), and of aphidicolin-synchronized Jurkat cells released from the G1 block for the indicated number of hours (bottom row; sync.). (C) Immunoprecipitation and western blot were performed as in (A) of lysates from the cell cycle synchronized cells shown in (B, bottom row). (D) Jurkat cells were infected with NL4-3 e-n-GFP (RT+) derivatives containing either wild-type Vpr and Vif (lane 1), Vpr but no Vif (lane 2), neither Vpr nor Vif (lane 3), or R80A mutant Vpr and no Vif (lane 4). Two days post-infection cells were lysed, immunoprecipitated, and immunoblotted as in (A) (top). Flow cytometric DNA content analysis was performed at the time of harvest and shown for the GFP+ (HIV-infected) population of each sample (bottom, numbering corresponds to lane numbers of blots). GFP expression is shown as an inset with the percentage of cells in the GFP-positive gate indicated within the plot.

    Article Snippet: Antibodies used with corresponding catalog numbers are as follows: β-actin (Sigma; A5441), Cdc2 (Santa Cruz; sc-54), Phospho-Tyr15-Cdc2 (Cell Signaling; 9111), Cdc25C (BD Pharmingen; 550922), Cdc25C (Santa Cruz; sc-327), Cdc25C (Calbiochem; CC26; EMSA), Phospho-Ser216-Cdc25C (Cell Signaling; 4901), Centrin (Sigma; C7736), CyclinB1 (Santa Cruz; sc-752 or sc-245), GAPDH (Abcam; ab9484), PARP (BD Transduction Laboratories; P76420), importin β (Sigma; I2534) PKA (Santa Cruz; sc-903), Plk1 (Upstate; 05–844), γ-Tubulin (Sigma; T3559); HIV-1 Vif (NIH ARRRP; 6459), HIV-1 Vpr (gift of K. Strebel, NIAID), 14-3-3 β (Santa Cruz; sc-25276), 14-3-3 θ (Santa Cruz; sc-732), 14-3-3 γ (Santa Cruz; sc-731c), 14-3-3 ζ (Santa Cruz; sc-1019; cross-reacts with β and σ isoforms).

    Techniques: Mutagenesis, Infection, Immunoprecipitation, Blocking Assay, Western Blot, Expressing

    Reduced 14-3-3 θ association with centrosomal proteins, centrin and Plk1, during HIV-1 infection-induced G 2 ,M arrest. (A) Jurkat cells were mock-infected (-) or infected with NL4-3 e-n-GFP derivatives encoding wild-type Vif and Vpr (wt), deleted Vpr (r-), deleted Vif (f-), or double deletion of Vpr and Vif (fr-) at an MOI of 1.5. Lysates were harvested two days post-infection and immunoprecipitated as in Fig. 2 (IP: 14-3-3 θ). Whole cell lysates (input) and IP samples were blotted for Plk1, 14-3-3 θ, centrin, Vif, and Vpr. There appeared to be poor transfer of centrin protein at the left edge of the gel (input lanes). The reduced band intensity for this sample does not reflect decreased centrin abundance as it was well-represented in the IP and similar experiments showed no changes in centrin expression upon HIV-1 infection. (B) DNA content analysis of the samples in (A) by propidium iodide staining. HIV-infected samples were pre-gated on GFP+ cells for DNA analysis. (C) Viability (large plot) and GFP expression by viable cells (inset) for samples in (A) and (B) were measured by flow cytometric detection of propidium iodide (PI) negative, large (high forward scatter) cells and GFP fluorescence (inset histogram), respectively, at the time lysates were harvested. Plots correspond to samples directly above in (B). The gates demarcate viable and GFP-positive cell populations and the percentage of cells within each gate is indicated. (D) Jurkat cells were mock-infected (m), infected with Vpr v as in (A), or infected with NL4-3 e-n-GFP (HIV) and harvested after 40 hours for immunoprecipitation with 14-3-3 θ and immunoblotting with importin β, CyclinB1, 14-3-3 θ, and Vpr. (E) DNA content analysis performed as in (B) is shown for the samples in (D). The inset histogram depicts the percentage of GFP+ cells expressing the NL4-3 e-n-GFP provirus. The DNA analysis for the HIV-infected sample was performed on the gated GFP population indicated. (F) Jurkat cells were mock-infected (m), infected with RT- NL4-3 e-n-GFP virions (Vpr v ) to deliver Vpr protein, or treated with adriamycin (adr). Cell lysates (input) were harvested after 40 hours for immunoprecipitation with importin β (IP) and immunoblotting with importin β, CyclinB1, Plk1, Cdk1, and Vpr as indicated. (G) Cell cycle analysis is shown for the samples in (F) at the time of lysis as measured by propidium iodide DNA staining as in (B).

    Journal: Biology Direct

    Article Title: 14-3-3 theta binding to cell cycle regulatory factors is enhanced by HIV-1 Vpr

    doi: 10.1186/1745-6150-3-17

    Figure Lengend Snippet: Reduced 14-3-3 θ association with centrosomal proteins, centrin and Plk1, during HIV-1 infection-induced G 2 ,M arrest. (A) Jurkat cells were mock-infected (-) or infected with NL4-3 e-n-GFP derivatives encoding wild-type Vif and Vpr (wt), deleted Vpr (r-), deleted Vif (f-), or double deletion of Vpr and Vif (fr-) at an MOI of 1.5. Lysates were harvested two days post-infection and immunoprecipitated as in Fig. 2 (IP: 14-3-3 θ). Whole cell lysates (input) and IP samples were blotted for Plk1, 14-3-3 θ, centrin, Vif, and Vpr. There appeared to be poor transfer of centrin protein at the left edge of the gel (input lanes). The reduced band intensity for this sample does not reflect decreased centrin abundance as it was well-represented in the IP and similar experiments showed no changes in centrin expression upon HIV-1 infection. (B) DNA content analysis of the samples in (A) by propidium iodide staining. HIV-infected samples were pre-gated on GFP+ cells for DNA analysis. (C) Viability (large plot) and GFP expression by viable cells (inset) for samples in (A) and (B) were measured by flow cytometric detection of propidium iodide (PI) negative, large (high forward scatter) cells and GFP fluorescence (inset histogram), respectively, at the time lysates were harvested. Plots correspond to samples directly above in (B). The gates demarcate viable and GFP-positive cell populations and the percentage of cells within each gate is indicated. (D) Jurkat cells were mock-infected (m), infected with Vpr v as in (A), or infected with NL4-3 e-n-GFP (HIV) and harvested after 40 hours for immunoprecipitation with 14-3-3 θ and immunoblotting with importin β, CyclinB1, 14-3-3 θ, and Vpr. (E) DNA content analysis performed as in (B) is shown for the samples in (D). The inset histogram depicts the percentage of GFP+ cells expressing the NL4-3 e-n-GFP provirus. The DNA analysis for the HIV-infected sample was performed on the gated GFP population indicated. (F) Jurkat cells were mock-infected (m), infected with RT- NL4-3 e-n-GFP virions (Vpr v ) to deliver Vpr protein, or treated with adriamycin (adr). Cell lysates (input) were harvested after 40 hours for immunoprecipitation with importin β (IP) and immunoblotting with importin β, CyclinB1, Plk1, Cdk1, and Vpr as indicated. (G) Cell cycle analysis is shown for the samples in (F) at the time of lysis as measured by propidium iodide DNA staining as in (B).

    Article Snippet: Antibodies used with corresponding catalog numbers are as follows: β-actin (Sigma; A5441), Cdc2 (Santa Cruz; sc-54), Phospho-Tyr15-Cdc2 (Cell Signaling; 9111), Cdc25C (BD Pharmingen; 550922), Cdc25C (Santa Cruz; sc-327), Cdc25C (Calbiochem; CC26; EMSA), Phospho-Ser216-Cdc25C (Cell Signaling; 4901), Centrin (Sigma; C7736), CyclinB1 (Santa Cruz; sc-752 or sc-245), GAPDH (Abcam; ab9484), PARP (BD Transduction Laboratories; P76420), importin β (Sigma; I2534) PKA (Santa Cruz; sc-903), Plk1 (Upstate; 05–844), γ-Tubulin (Sigma; T3559); HIV-1 Vif (NIH ARRRP; 6459), HIV-1 Vpr (gift of K. Strebel, NIAID), 14-3-3 β (Santa Cruz; sc-25276), 14-3-3 θ (Santa Cruz; sc-732), 14-3-3 γ (Santa Cruz; sc-731c), 14-3-3 ζ (Santa Cruz; sc-1019; cross-reacts with β and σ isoforms).

    Techniques: Infection, Immunoprecipitation, Expressing, Staining, Fluorescence, Western Blot, Cell Cycle Assay, Lysis

    Cell cycle regulatory protein binding to 14-3-3 θ is also enhanced during G 2 ,M arrest induced by adriamycin. Jurkat T cells were treated with adriamycin (Adr; 0.2 μg/ml) for 24, 48, or 72 hours (h) or untreated (0) and examined for 14-3-3 θ co-immunoprecipitating (IP) proteins. (A) Western blot analysis of Plk1, CyclinB1, Cdk1, 14-3-3 θ and centrin in whole cell lysates before IP (input) and after 14-3-3 θ IP (IP: 14-3-3 θ) at the time of adriamycin treatment indicated. These data are representative of three independent experiments. Although the amount of 14-3-3 θ appears less in the untreated (0) time point of the IP, this was not a reproducible finding. (B) Viability of the samples used in the IP in (A) determined by flow cytometry detection of propidium iodide (PI) exclusion and large size (high forward scatter). The percentage of viable cells is indicated. Flow cytometric histograms of the DNA content for each sample determined by propidium iodide DNA staining (x-axis) are shown as an inset.

    Journal: Biology Direct

    Article Title: 14-3-3 theta binding to cell cycle regulatory factors is enhanced by HIV-1 Vpr

    doi: 10.1186/1745-6150-3-17

    Figure Lengend Snippet: Cell cycle regulatory protein binding to 14-3-3 θ is also enhanced during G 2 ,M arrest induced by adriamycin. Jurkat T cells were treated with adriamycin (Adr; 0.2 μg/ml) for 24, 48, or 72 hours (h) or untreated (0) and examined for 14-3-3 θ co-immunoprecipitating (IP) proteins. (A) Western blot analysis of Plk1, CyclinB1, Cdk1, 14-3-3 θ and centrin in whole cell lysates before IP (input) and after 14-3-3 θ IP (IP: 14-3-3 θ) at the time of adriamycin treatment indicated. These data are representative of three independent experiments. Although the amount of 14-3-3 θ appears less in the untreated (0) time point of the IP, this was not a reproducible finding. (B) Viability of the samples used in the IP in (A) determined by flow cytometry detection of propidium iodide (PI) exclusion and large size (high forward scatter). The percentage of viable cells is indicated. Flow cytometric histograms of the DNA content for each sample determined by propidium iodide DNA staining (x-axis) are shown as an inset.

    Article Snippet: Antibodies used with corresponding catalog numbers are as follows: β-actin (Sigma; A5441), Cdc2 (Santa Cruz; sc-54), Phospho-Tyr15-Cdc2 (Cell Signaling; 9111), Cdc25C (BD Pharmingen; 550922), Cdc25C (Santa Cruz; sc-327), Cdc25C (Calbiochem; CC26; EMSA), Phospho-Ser216-Cdc25C (Cell Signaling; 4901), Centrin (Sigma; C7736), CyclinB1 (Santa Cruz; sc-752 or sc-245), GAPDH (Abcam; ab9484), PARP (BD Transduction Laboratories; P76420), importin β (Sigma; I2534) PKA (Santa Cruz; sc-903), Plk1 (Upstate; 05–844), γ-Tubulin (Sigma; T3559); HIV-1 Vif (NIH ARRRP; 6459), HIV-1 Vpr (gift of K. Strebel, NIAID), 14-3-3 β (Santa Cruz; sc-25276), 14-3-3 θ (Santa Cruz; sc-732), 14-3-3 γ (Santa Cruz; sc-731c), 14-3-3 ζ (Santa Cruz; sc-1019; cross-reacts with β and σ isoforms).

    Techniques: Protein Binding, Western Blot, Flow Cytometry, Staining

    Cell cycle regulatory proteins reside in the centrosome during G 2 ,M arrest induced by HIV-1 infection and adriamycin. (A) Western blot analysis of centrosomes isolated from Jurkat cells infected with NL4-3 e-n-GFP (HIV; MOI of 2) for two days. Centrosomes were isolated by discontinuous sucrose gradient and fractions were collected and separated by SDS-PAGE and western blotted for Plk1, CyclinB1, γ-tubulin, Cdk1, 14-3-3 θ, centrin, Vif, and Vpr. Lanes 1–6 represent fractions from the bottom of the gradient upward, with centrosomes most abundant in lane 3. Whole cell lysates were run in lane 8 and volume from the top of the gradient equivalent to that used for each fraction was run in lane 7 to demonstrate sedimentation of centrosomal proteins through the gradient.

    Journal: Biology Direct

    Article Title: 14-3-3 theta binding to cell cycle regulatory factors is enhanced by HIV-1 Vpr

    doi: 10.1186/1745-6150-3-17

    Figure Lengend Snippet: Cell cycle regulatory proteins reside in the centrosome during G 2 ,M arrest induced by HIV-1 infection and adriamycin. (A) Western blot analysis of centrosomes isolated from Jurkat cells infected with NL4-3 e-n-GFP (HIV; MOI of 2) for two days. Centrosomes were isolated by discontinuous sucrose gradient and fractions were collected and separated by SDS-PAGE and western blotted for Plk1, CyclinB1, γ-tubulin, Cdk1, 14-3-3 θ, centrin, Vif, and Vpr. Lanes 1–6 represent fractions from the bottom of the gradient upward, with centrosomes most abundant in lane 3. Whole cell lysates were run in lane 8 and volume from the top of the gradient equivalent to that used for each fraction was run in lane 7 to demonstrate sedimentation of centrosomal proteins through the gradient. "L" indicates a light exposure and "D" indicates a darker exposure of the chemilumigraph of the middle part of the gel. (B) Viability (top) and DNA content analysis (bottom) by flow cytometry for the culture in (A; HIV (+)) and untreated Jurkats without (-) HIV infection. The percentage of viable cells was determined by propidium iodide (PI) exclusion and high forward scatter and is indicated in the lower right corner. Infection efficiency was measured by GFP expression (inset; gated population) and the percentage is indicated. DNA content was measured by flow cytometric detection of DNA stained with propidium iodide. The GFP-positive population was analyzed for the HIV-infected culture. (C) Jurkat T cells were treated with adriamycin (adr; 0.2 μg/ml; right panel) for two days or grown asynchronously (left panel) and centrosomes were isolated by discontinuous sucrose gradients as in (A). Centrosomes were most abundant in lanes 2–3 (untreated cells) or lanes 3–4 (adriamycin treated cells). Lanes were as described in panel A. (D) Cell cycle flow cytometric analysis of Jurkat cells in (C).

    Article Snippet: Antibodies used with corresponding catalog numbers are as follows: β-actin (Sigma; A5441), Cdc2 (Santa Cruz; sc-54), Phospho-Tyr15-Cdc2 (Cell Signaling; 9111), Cdc25C (BD Pharmingen; 550922), Cdc25C (Santa Cruz; sc-327), Cdc25C (Calbiochem; CC26; EMSA), Phospho-Ser216-Cdc25C (Cell Signaling; 4901), Centrin (Sigma; C7736), CyclinB1 (Santa Cruz; sc-752 or sc-245), GAPDH (Abcam; ab9484), PARP (BD Transduction Laboratories; P76420), importin β (Sigma; I2534) PKA (Santa Cruz; sc-903), Plk1 (Upstate; 05–844), γ-Tubulin (Sigma; T3559); HIV-1 Vif (NIH ARRRP; 6459), HIV-1 Vpr (gift of K. Strebel, NIAID), 14-3-3 β (Santa Cruz; sc-25276), 14-3-3 θ (Santa Cruz; sc-732), 14-3-3 γ (Santa Cruz; sc-731c), 14-3-3 ζ (Santa Cruz; sc-1019; cross-reacts with β and σ isoforms).

    Techniques: Infection, Western Blot, Isolation, SDS Page, Sedimentation, Flow Cytometry, Expressing, Staining

    A, FACS analyses showed that the sub-G1 phase HuH7 cell population increased from approximately 0.7% to 7.7%, and cells in the G1 phase increased from approximately 69% to 87% 48 h after TGF-β1 treatment. Data represent the means ± S.D. of three experiments. * indicates significant differences between each group (P<0.05). B, Cdc2 kinase activity was determined based on the level of phosphorylated histone H1 using histone H1 as a substrate. Cdc2 was activated 48 h after TGF-β1 treatment in HuH7 cells. However, cdc2 was not activated in HuH7R cells, which were isolated as an apoptosis-resistant clone from TGF-β1-treated HuH7 cells. Roscovitine (Ros)-pretreated HuH7 cells did not show cdc2 activation. A representative image from three independent experiments is shown. C, After TGF-β1 treatment (48 h), we observed cdc2 Tyr15 dephosphorylation in association with Wee1 kinase down-regulation in apoptotic cells. Pretreatment with 20 µM roscovitine completely abolished apoptosis and restored Wee1 kinase expression. TGF-β1 treatment induced G1 cell cycle arrest in HuH7R cells; however, Wee1 kinase expression and non-phosphorylated cdc2 Tyr 15 were similar to those of roscovitine-pretreated HuH7 cells. A representative image of three experiments is shown. D, Wee1 down-regulation and cdc2 Tyr15 dephosphorylation commenced approximately 24 h after TGF-β1 treatment, which was similar to thea initiation of apoptosis. A representative Western blot image is shown in the upper panels. The results in the lower graphs represent the means ± S.D. of three experiments. * indicates significant differences between each group (P<0.05).

    Journal: PLoS ONE

    Article Title: Efficient Induction of Apoptosis by Wee1 Kinase Inhibition in Hepatocellular Carcinoma Cells

    doi: 10.1371/journal.pone.0100495

    Figure Lengend Snippet: A, FACS analyses showed that the sub-G1 phase HuH7 cell population increased from approximately 0.7% to 7.7%, and cells in the G1 phase increased from approximately 69% to 87% 48 h after TGF-β1 treatment. Data represent the means ± S.D. of three experiments. * indicates significant differences between each group (P<0.05). B, Cdc2 kinase activity was determined based on the level of phosphorylated histone H1 using histone H1 as a substrate. Cdc2 was activated 48 h after TGF-β1 treatment in HuH7 cells. However, cdc2 was not activated in HuH7R cells, which were isolated as an apoptosis-resistant clone from TGF-β1-treated HuH7 cells. Roscovitine (Ros)-pretreated HuH7 cells did not show cdc2 activation. A representative image from three independent experiments is shown. C, After TGF-β1 treatment (48 h), we observed cdc2 Tyr15 dephosphorylation in association with Wee1 kinase down-regulation in apoptotic cells. Pretreatment with 20 µM roscovitine completely abolished apoptosis and restored Wee1 kinase expression. TGF-β1 treatment induced G1 cell cycle arrest in HuH7R cells; however, Wee1 kinase expression and non-phosphorylated cdc2 Tyr 15 were similar to those of roscovitine-pretreated HuH7 cells. A representative image of three experiments is shown. D, Wee1 down-regulation and cdc2 Tyr15 dephosphorylation commenced approximately 24 h after TGF-β1 treatment, which was similar to thea initiation of apoptosis. A representative Western blot image is shown in the upper panels. The results in the lower graphs represent the means ± S.D. of three experiments. * indicates significant differences between each group (P<0.05).

    Article Snippet: Immunoblots were prepared as described previously and probed with anti-Wee1, anti-β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), or anti-cdc2-phospho-Tyr15 (Cell Signaling Technology, Danvers, MA, USA).

    Techniques: Activity Assay, Isolation, Activation Assay, De-Phosphorylation Assay, Expressing, Western Blot

    (A) Flow cytometric determination of DNA content was analyzed using the Accuri C6 Flow Cytometer. shLASP-1-transfected HSC-3 and Ca9-22 cells show significant (* P <0.025; Mann-Whitney U test with Bonferroni correction) G2 phase accumulation as opposed to the shMock-transfected cells. (B) Immunoblotting analysis shows down-regulation of cyclin A and cyclin B and up-regulation of phospho-cdc2 in the knockdown cells.

    Journal: PLoS ONE

    Article Title: Overexpression of LIM and SH3 Protein 1 Leading to Accelerated G2/M Phase Transition Contributes to Enhanced Tumourigenesis in Oral Cancer

    doi: 10.1371/journal.pone.0083187

    Figure Lengend Snippet: (A) Flow cytometric determination of DNA content was analyzed using the Accuri C6 Flow Cytometer. shLASP-1-transfected HSC-3 and Ca9-22 cells show significant (* P <0.025; Mann-Whitney U test with Bonferroni correction) G2 phase accumulation as opposed to the shMock-transfected cells. (B) Immunoblotting analysis shows down-regulation of cyclin A and cyclin B and up-regulation of phospho-cdc2 in the knockdown cells.

    Article Snippet: Protein extracts were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis in 10% gel and transferred to nitrocellulose membranes before incubation overnight with primary antibodies against LASP-1 (Applied Biological Materials, Richmond, BC, Canada), cyclin A, cyclin B, or phospho-cdc2 (Tyr15) (Cell Signaling Technology, Danvers, MA) at 4°C.

    Techniques: Flow Cytometry, Transfection, MANN-WHITNEY, Western Blot