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: 96/100, 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 96 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    polyclonal anti phospho cdc2 tyr15 - by Bioz Stars, 2023-03
    96/100 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)


    Bioz Verified Symbol Cell Signaling Technology Inc is a verified supplier
    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
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    Structured Review

    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: 96/100, 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 96 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    polyclonal anti phospho cdc2 tyr15 - by Bioz Stars, 2023-03
    96/100 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

    rabbit polyclonal antibody against 15 tyr  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit polyclonal antibody against 15 tyr
    Rabbit Polyclonal Antibody Against 15 Tyr, 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/rabbit polyclonal antibody against 15 tyr/product/Cell Signaling Technology Inc
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit polyclonal antibody against 15 tyr - by Bioz Stars, 2023-03
    97/100 stars

    Images

    rabbit polyclonal anti phospho cdc2 tyr15  (Cell Signaling Technology Inc)


    Bioz Verified Symbol Cell Signaling Technology Inc is a verified supplier
    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
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    Structured Review

    Cell Signaling Technology Inc rabbit polyclonal anti phospho cdc2 tyr15
    (A-D) Two hours after release from a double thymidine block (DTB), synchronized HeLa S3 cells were treated with MeOH or the indicated concentrations of Pla-B. Black triangles indicate the time points of cell harvest and sample preparation (A). Cell cycle was analyzed at the indicated time points by cytometry (B). Morphology of the cells was observed under a microscope and round cells were counted at the indicated time points (C). Protein samples were prepared at the indicated time points. The protein levels of indicated proteins and phosphorylation status of <t>Cdk1</t> were analysed by immunoblotting. Protein levels of α-tubulin were analysed as an internal control (D). Error bars indicate s.d. (n = 3).
    Rabbit 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/rabbit 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
    rabbit polyclonal anti phospho cdc2 tyr15 - by Bioz Stars, 2023-03
    97/100 stars

    Images

    1) Product Images from "A truncated form of the p27 CDK inhibitor translated from pre-mRNA causes cell cycle arrest at G2 phase"

    Article Title: A truncated form of the p27 CDK inhibitor translated from pre-mRNA causes cell cycle arrest at G2 phase

    Journal: bioRxiv

    doi: 10.1101/2022.01.12.476115

    (A-D) Two hours after release from a double thymidine block (DTB), synchronized HeLa S3 cells were treated with MeOH or the indicated concentrations of Pla-B. Black triangles indicate the time points of cell harvest and sample preparation (A). Cell cycle was analyzed at the indicated time points by cytometry (B). Morphology of the cells was observed under a microscope and round cells were counted at the indicated time points (C). Protein samples were prepared at the indicated time points. The protein levels of indicated proteins and phosphorylation status of Cdk1 were analysed by immunoblotting. Protein levels of α-tubulin were analysed as an internal control (D). Error bars indicate s.d. (n = 3).
    Figure Legend Snippet: (A-D) Two hours after release from a double thymidine block (DTB), synchronized HeLa S3 cells were treated with MeOH or the indicated concentrations of Pla-B. Black triangles indicate the time points of cell harvest and sample preparation (A). Cell cycle was analyzed at the indicated time points by cytometry (B). Morphology of the cells was observed under a microscope and round cells were counted at the indicated time points (C). Protein samples were prepared at the indicated time points. The protein levels of indicated proteins and phosphorylation status of Cdk1 were analysed by immunoblotting. Protein levels of α-tubulin were analysed as an internal control (D). Error bars indicate s.d. (n = 3).

    Techniques Used: Blocking Assay, Sample Prep, Cytometry, Microscopy, Western Blot

    (A) Cells expressing Flag-p27* under the control of tetracycline were synchronized by a double thymidine block. The cells were treated with 1 μg/ml DOX at the same time as release from the double thymidine block. The cells were harvested at 8 h after release from the double thymidine block (G2/M phase) and then immunoprecipitation was performed using anti-DDDDK (Flag) antibodies. Flag-tagged and coimmunoprecipitated proteins were analysed by immunoblotting. (B, C) Purified Flag-tagged proteins were applied to an in vitro kinase assay reaction and kinase activities of Cyclin A2/Cdk1 (B) and Cyclin B1/Cdk1 (C) were measured. Statistical significance was assessed by the one-way ANOVA and Dunnett’s test (*: P < 0.05; **: P < 0.01; ***: P < 0.01). Error bars indicate s.d. (n = 3).
    Figure Legend Snippet: (A) Cells expressing Flag-p27* under the control of tetracycline were synchronized by a double thymidine block. The cells were treated with 1 μg/ml DOX at the same time as release from the double thymidine block. The cells were harvested at 8 h after release from the double thymidine block (G2/M phase) and then immunoprecipitation was performed using anti-DDDDK (Flag) antibodies. Flag-tagged and coimmunoprecipitated proteins were analysed by immunoblotting. (B, C) Purified Flag-tagged proteins were applied to an in vitro kinase assay reaction and kinase activities of Cyclin A2/Cdk1 (B) and Cyclin B1/Cdk1 (C) were measured. Statistical significance was assessed by the one-way ANOVA and Dunnett’s test (*: P < 0.05; **: P < 0.01; ***: P < 0.01). Error bars indicate s.d. (n = 3).

    Techniques Used: Expressing, Blocking Assay, Immunoprecipitation, Western Blot, Purification, In Vitro, Kinase Assay

    rabbit polyclonal phospho cdc2 tyr15  (Cell Signaling Technology Inc)


    Bioz Verified Symbol Cell Signaling Technology Inc is a verified supplier
    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
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    Structured Review

    Cell Signaling Technology Inc rabbit polyclonal phospho cdc2 tyr15
    A DSG (red) and the KKR (violet) motifs, and the triple K36A/K38A/R40A (KKR/AAA) Xenopus Arpp19 alanine mutant. Table depicting dephosphorylation time, B55-Arpp19 interaction and the capacity to promote mitotic entry in Arpp19-depleted extracts. B 50 ng of wild type or KKR/AAA Arpp19 mutant phosphorylated ‘in vitro’ by GwlK72M was supplemented to kinase-inactivated extracts depleted or not of the B55 protein. Arpp19 levels and of S67/S71 phosphorylation were analysed by western blotting and autoradiography, respectively. The 33 P-Arpp19/western blotting Arpp19 signal ratios were calculated using ImageJ for each time point. The percentage of the ratio remaining at each time point with respect to the ratio at 0 min was calculated and represented in bar graphs as the mean percentage ± SD; n = 3 biological independent samples. C A His-Arpp19 pulldown equivalent to 20 ng of wild type or KKR/AAA mutant was submitted to western blotting, and the amount of B55 and the levels of Arpp19 bound to the beads shown. B55/Arpp19 signal ratios were calculated using ImageJ and represented in a bar graph as the mean ratio ± SD; n = 5 biological independent samples. D Arpp19-depleted extracts were supplemented with human GwlK72M and a wild type or a KKR/AAA Arpp19 mutant and phosphorylation of Human Gwl, of <t>Tyr15,</t> of <t>Cdk1</t> and Arpp19 ectopic levels (His-Arpp19) were assessed. E A schematic of DSG regions indicating residues mutated into alanine or threonine. Table representing results on the S67/S71 dephosphorylation time and the capacities to bind B55 or to restore mitotic entry in Arpp19-depleted egg extracts of each Arpp19 form. F Wild-type Arpp19 and the indicated mutants of the DSG motif were ‘in vitro’ phosphorylated by hGwlK72M and 1 µl sample removed at 10 and 40 min, to measure S67/S71 phosphorylation by autoradiography ( P33 Arp). The amount of Arpp19 was assessed by Coomassie blue staining (Arp) 33 . P-Arpp19 levels were normalized by Arpp19 amount using Coomassie blue signal and the increase in phosphorylation between time 10 and 40 min calculated. Data from three different experiments was then used to obtain the mean ± SD and represented in a bar graph; n = 3 biological independent samples.
    Rabbit Polyclonal 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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    Images

    1) Product Images from "The study of the determinants controlling Arpp19 phosphatase-inhibitory activity reveals an Arpp19/PP2A-B55 feedback loop"

    Article Title: The study of the determinants controlling Arpp19 phosphatase-inhibitory activity reveals an Arpp19/PP2A-B55 feedback loop

    Journal: Nature Communications

    doi: 10.1038/s41467-021-23657-0

    A DSG (red) and the KKR (violet) motifs, and the triple K36A/K38A/R40A (KKR/AAA) Xenopus Arpp19 alanine mutant. Table depicting dephosphorylation time, B55-Arpp19 interaction and the capacity to promote mitotic entry in Arpp19-depleted extracts. B 50 ng of wild type or KKR/AAA Arpp19 mutant phosphorylated ‘in vitro’ by GwlK72M was supplemented to kinase-inactivated extracts depleted or not of the B55 protein. Arpp19 levels and of S67/S71 phosphorylation were analysed by western blotting and autoradiography, respectively. The 33 P-Arpp19/western blotting Arpp19 signal ratios were calculated using ImageJ for each time point. The percentage of the ratio remaining at each time point with respect to the ratio at 0 min was calculated and represented in bar graphs as the mean percentage ± SD; n = 3 biological independent samples. C A His-Arpp19 pulldown equivalent to 20 ng of wild type or KKR/AAA mutant was submitted to western blotting, and the amount of B55 and the levels of Arpp19 bound to the beads shown. B55/Arpp19 signal ratios were calculated using ImageJ and represented in a bar graph as the mean ratio ± SD; n = 5 biological independent samples. D Arpp19-depleted extracts were supplemented with human GwlK72M and a wild type or a KKR/AAA Arpp19 mutant and phosphorylation of Human Gwl, of Tyr15, of Cdk1 and Arpp19 ectopic levels (His-Arpp19) were assessed. E A schematic of DSG regions indicating residues mutated into alanine or threonine. Table representing results on the S67/S71 dephosphorylation time and the capacities to bind B55 or to restore mitotic entry in Arpp19-depleted egg extracts of each Arpp19 form. F Wild-type Arpp19 and the indicated mutants of the DSG motif were ‘in vitro’ phosphorylated by hGwlK72M and 1 µl sample removed at 10 and 40 min, to measure S67/S71 phosphorylation by autoradiography ( P33 Arp). The amount of Arpp19 was assessed by Coomassie blue staining (Arp) 33 . P-Arpp19 levels were normalized by Arpp19 amount using Coomassie blue signal and the increase in phosphorylation between time 10 and 40 min calculated. Data from three different experiments was then used to obtain the mean ± SD and represented in a bar graph; n = 3 biological independent samples.
    Figure Legend Snippet: A DSG (red) and the KKR (violet) motifs, and the triple K36A/K38A/R40A (KKR/AAA) Xenopus Arpp19 alanine mutant. Table depicting dephosphorylation time, B55-Arpp19 interaction and the capacity to promote mitotic entry in Arpp19-depleted extracts. B 50 ng of wild type or KKR/AAA Arpp19 mutant phosphorylated ‘in vitro’ by GwlK72M was supplemented to kinase-inactivated extracts depleted or not of the B55 protein. Arpp19 levels and of S67/S71 phosphorylation were analysed by western blotting and autoradiography, respectively. The 33 P-Arpp19/western blotting Arpp19 signal ratios were calculated using ImageJ for each time point. The percentage of the ratio remaining at each time point with respect to the ratio at 0 min was calculated and represented in bar graphs as the mean percentage ± SD; n = 3 biological independent samples. C A His-Arpp19 pulldown equivalent to 20 ng of wild type or KKR/AAA mutant was submitted to western blotting, and the amount of B55 and the levels of Arpp19 bound to the beads shown. B55/Arpp19 signal ratios were calculated using ImageJ and represented in a bar graph as the mean ratio ± SD; n = 5 biological independent samples. D Arpp19-depleted extracts were supplemented with human GwlK72M and a wild type or a KKR/AAA Arpp19 mutant and phosphorylation of Human Gwl, of Tyr15, of Cdk1 and Arpp19 ectopic levels (His-Arpp19) were assessed. E A schematic of DSG regions indicating residues mutated into alanine or threonine. Table representing results on the S67/S71 dephosphorylation time and the capacities to bind B55 or to restore mitotic entry in Arpp19-depleted egg extracts of each Arpp19 form. F Wild-type Arpp19 and the indicated mutants of the DSG motif were ‘in vitro’ phosphorylated by hGwlK72M and 1 µl sample removed at 10 and 40 min, to measure S67/S71 phosphorylation by autoradiography ( P33 Arp). The amount of Arpp19 was assessed by Coomassie blue staining (Arp) 33 . P-Arpp19 levels were normalized by Arpp19 amount using Coomassie blue signal and the increase in phosphorylation between time 10 and 40 min calculated. Data from three different experiments was then used to obtain the mean ± SD and represented in a bar graph; n = 3 biological independent samples.

    Techniques Used: Mutagenesis, De-Phosphorylation Assay, In Vitro, Western Blot, Autoradiography, Staining

    A B55 levels associated to 20 ng of wild type or the indicated DSG mutants of His-Arpp19-pulldowns. Arpp19 amount in these pulldowns is also shown. Data were represented as mean B55/Arpp19 ratio ± SD. Two-tailed unpaired Student’s t -tests were performed in each pulldown to determine statistical relevance. p vs. wild-type Arpp19 is shown; n = 3 biological independent samples for mutants Y68A, G72A and Y74A; n = 5 for the D73A, n = 6 for the wild-type form and n = 4 for the rest. B The wild type and the indicated DSG mutant forms of Arpp19 were thio-phosphorylated and used for His-pulldown. B55 and Arpp19 levels were checked by western blotting and shown. The B55/Arpp19 ratios were quantified and represented in a bar graph as mean ± SD. Two-tailed unpaired Student t -tests were performed in each pulldown to determine statistical relevance. p vs. wild-type Arpp19 is shown; n = 3 biological independent samples. C The B55/Arpp19 ratios were obtained as in A for the indicated mutants and represented in a bar graph as mean ± SD. Two-tailed unpaired; p vs. wild-type Arpp19 is shown; n = 3 biological independent samples for the wild-type form; n = 8 for the G72A mutant, n = 5 for the G72A-S71A mutant and n = 3 for the G72A-S71T mutant. D Prophase oocytes were injected or not (PG) with 50 ng of the wild-type His-Arpp19 protein or with the indicated mutant forms and, 1 h later, treated with progesterone. GVBD was then scored as a function of time. Germinal vesicle (GV) and mature oocytes (GVBD) were western blotted to determine the levels of the injected protein, as well as the phosphorylation of Gwl and of the inhibitory site of Cdk1 Tyrosine 15. E As for E , except that the indicated mutant form of Arpp19 was used.
    Figure Legend Snippet: A B55 levels associated to 20 ng of wild type or the indicated DSG mutants of His-Arpp19-pulldowns. Arpp19 amount in these pulldowns is also shown. Data were represented as mean B55/Arpp19 ratio ± SD. Two-tailed unpaired Student’s t -tests were performed in each pulldown to determine statistical relevance. p vs. wild-type Arpp19 is shown; n = 3 biological independent samples for mutants Y68A, G72A and Y74A; n = 5 for the D73A, n = 6 for the wild-type form and n = 4 for the rest. B The wild type and the indicated DSG mutant forms of Arpp19 were thio-phosphorylated and used for His-pulldown. B55 and Arpp19 levels were checked by western blotting and shown. The B55/Arpp19 ratios were quantified and represented in a bar graph as mean ± SD. Two-tailed unpaired Student t -tests were performed in each pulldown to determine statistical relevance. p vs. wild-type Arpp19 is shown; n = 3 biological independent samples. C The B55/Arpp19 ratios were obtained as in A for the indicated mutants and represented in a bar graph as mean ± SD. Two-tailed unpaired; p vs. wild-type Arpp19 is shown; n = 3 biological independent samples for the wild-type form; n = 8 for the G72A mutant, n = 5 for the G72A-S71A mutant and n = 3 for the G72A-S71T mutant. D Prophase oocytes were injected or not (PG) with 50 ng of the wild-type His-Arpp19 protein or with the indicated mutant forms and, 1 h later, treated with progesterone. GVBD was then scored as a function of time. Germinal vesicle (GV) and mature oocytes (GVBD) were western blotted to determine the levels of the injected protein, as well as the phosphorylation of Gwl and of the inhibitory site of Cdk1 Tyrosine 15. E As for E , except that the indicated mutant form of Arpp19 was used.

    Techniques Used: Two Tailed Test, Mutagenesis, Western Blot, Injection

    A Wild type and S109/S113D Arpp19 mutant were phosphorylated ‘in vitro’ with of [γ 33 P] ATP by GwlK72M on S67/S71 and supplemented to kinase-inactivated Xenopus egg extracts. The dephosphorylation of this residue was analysed at the indicated times by autoradiography ( P33 Arp) and the amount of Arpp19 in each sample measured by western blotting (Arp). B CytoStatic Factor (CSF) egg extracts were supplemented with a trace level of Arpp19-purified protein and activated to exit meiosis by the addition of active CamKII. The levels and dephosphorylation of the indicated proteins were analysed by western blotting, whereas Cyclin B/Cdk1 activity was measured by histone H1 phosphorylation (H1K). ‘Inter’ denotes interphase egg extracts. C As for C , except that S67/S71 Arpp19 dephosphorylation and PP2A-B55 reactivation upon meiosis exit in these extracts was blocked by the concomitant addition of GwlK72M-purified protein.
    Figure Legend Snippet: A Wild type and S109/S113D Arpp19 mutant were phosphorylated ‘in vitro’ with of [γ 33 P] ATP by GwlK72M on S67/S71 and supplemented to kinase-inactivated Xenopus egg extracts. The dephosphorylation of this residue was analysed at the indicated times by autoradiography ( P33 Arp) and the amount of Arpp19 in each sample measured by western blotting (Arp). B CytoStatic Factor (CSF) egg extracts were supplemented with a trace level of Arpp19-purified protein and activated to exit meiosis by the addition of active CamKII. The levels and dephosphorylation of the indicated proteins were analysed by western blotting, whereas Cyclin B/Cdk1 activity was measured by histone H1 phosphorylation (H1K). ‘Inter’ denotes interphase egg extracts. C As for C , except that S67/S71 Arpp19 dephosphorylation and PP2A-B55 reactivation upon meiosis exit in these extracts was blocked by the concomitant addition of GwlK72M-purified protein.

    Techniques Used: Mutagenesis, In Vitro, De-Phosphorylation Assay, Autoradiography, Western Blot, Purification, Activity Assay

    rabbit polyclonal anti phospho cdc2 tyr15  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit polyclonal anti phospho cdc2 tyr15
    Depletion of EML1 in oocytes impairs the activation of MPF. Dynamic changes in the levels of CCNB1, pCDK1-Y15, and <t>CDK1</t> in the oocytes during the period of oocytes meiosis resumption (A,B) and MI-to-MII transition (C,D) were assessed by Western Blot analysis, with the level of ACTB used as internal control. Lysate from 50 oocytes was loaded in each lane. The representative gel images of three independent experiments are shown in panels (A,C) , and the quantification of the Western Blot results are shown in panels (B,D) . * P < 0.05, compared with the Control-MO group.
    Rabbit 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
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    Images

    1) Product Images from "Echinoderm Microtubule Associated Protein Like 1 Is Indispensable for Oocyte Spindle Assembly and Meiotic Progression in Mice"

    Article Title: Echinoderm Microtubule Associated Protein Like 1 Is Indispensable for Oocyte Spindle Assembly and Meiotic Progression in Mice

    Journal: Frontiers in Cell and Developmental Biology

    doi: 10.3389/fcell.2021.687522

    Depletion of EML1 in oocytes impairs the activation of MPF. Dynamic changes in the levels of CCNB1, pCDK1-Y15, and CDK1 in the oocytes during the period of oocytes meiosis resumption (A,B) and MI-to-MII transition (C,D) were assessed by Western Blot analysis, with the level of ACTB used as internal control. Lysate from 50 oocytes was loaded in each lane. The representative gel images of three independent experiments are shown in panels (A,C) , and the quantification of the Western Blot results are shown in panels (B,D) . * P < 0.05, compared with the Control-MO group.
    Figure Legend Snippet: Depletion of EML1 in oocytes impairs the activation of MPF. Dynamic changes in the levels of CCNB1, pCDK1-Y15, and CDK1 in the oocytes during the period of oocytes meiosis resumption (A,B) and MI-to-MII transition (C,D) were assessed by Western Blot analysis, with the level of ACTB used as internal control. Lysate from 50 oocytes was loaded in each lane. The representative gel images of three independent experiments are shown in panels (A,C) , and the quantification of the Western Blot results are shown in panels (B,D) . * P < 0.05, compared with the Control-MO group.

    Techniques Used: Activation Assay, Western Blot

    Activation of MPF by inhibitors of WEE1/2 kinases rescues the defects of meiotic progression in EML1-knocked down oocytes. (A) Oocytes microinjected with Control-MO and EML1-MO were first maintained at GV stage for 20 h, and then transferred to maturation medium with (for EML1-MO group) or without (for Control-MO group) the supplementation of the inhibitor of WEE1/2 kinases, PD166285, to allow for maturation. Oocyte GVB was scored during the culture. * P < 0.05, compared with the Control-MO group. (B) Western Blot analysis of the changes in the levels of CCNB1, pCDK1-Y15, and CDK1 in the EML1-knocked down oocytes that were treated with PD166285. ACTB was used as the loading control. Lysate from 50 oocytes was loaded in each lane. The representative gel images of three independent experiments are shown. ( C–E) Assessment of the effect of PD166285 on the progression of meiosis to MII in EML1-knocked down oocytes. Oocytes microinjected with Control-MO and EML1-MO were first maintained at GV stage for 24 h. Then the EML1-MO oocytes were split into two groups, one of which was transferred to maturation medium supplemented with PD16628S and directly cultured for 20 h, while the other one was first transferred to maturation medium without PD166285 for 14 h, and then in the PD166285-supplemented medium for another 4 h culture. PBE, PN formation, and spindle morphology were then scored and analyzed. Representative images of the oocytes with the microtubules labeled with anti-tubulin antibody (green) and DNA stained with Hochest (blue) are shown in panel (C) . Scale Bar = 20 μm. Quantification of the percentages of oocytes with PB1 and PN, and those with normal MII-spindles are shown in panels (D,E) , respectively. * P < 0.05, ** P < 0.01, *** P < 0.005, compared with the EML1-MO group. ns denote no significant difference between the two groups compared.
    Figure Legend Snippet: Activation of MPF by inhibitors of WEE1/2 kinases rescues the defects of meiotic progression in EML1-knocked down oocytes. (A) Oocytes microinjected with Control-MO and EML1-MO were first maintained at GV stage for 20 h, and then transferred to maturation medium with (for EML1-MO group) or without (for Control-MO group) the supplementation of the inhibitor of WEE1/2 kinases, PD166285, to allow for maturation. Oocyte GVB was scored during the culture. * P < 0.05, compared with the Control-MO group. (B) Western Blot analysis of the changes in the levels of CCNB1, pCDK1-Y15, and CDK1 in the EML1-knocked down oocytes that were treated with PD166285. ACTB was used as the loading control. Lysate from 50 oocytes was loaded in each lane. The representative gel images of three independent experiments are shown. ( C–E) Assessment of the effect of PD166285 on the progression of meiosis to MII in EML1-knocked down oocytes. Oocytes microinjected with Control-MO and EML1-MO were first maintained at GV stage for 24 h. Then the EML1-MO oocytes were split into two groups, one of which was transferred to maturation medium supplemented with PD16628S and directly cultured for 20 h, while the other one was first transferred to maturation medium without PD166285 for 14 h, and then in the PD166285-supplemented medium for another 4 h culture. PBE, PN formation, and spindle morphology were then scored and analyzed. Representative images of the oocytes with the microtubules labeled with anti-tubulin antibody (green) and DNA stained with Hochest (blue) are shown in panel (C) . Scale Bar = 20 μm. Quantification of the percentages of oocytes with PB1 and PN, and those with normal MII-spindles are shown in panels (D,E) , respectively. * P < 0.05, ** P < 0.01, *** P < 0.005, compared with the EML1-MO group. ns denote no significant difference between the two groups compared.

    Techniques Used: Activation Assay, Western Blot, Cell Culture, Labeling, Staining

    anti human phospho sgk rabbit polyclonal antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti human phospho sgk rabbit polyclonal antibody
    Anti Human Phospho Sgk Rabbit Polyclonal Antibody, 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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    rabbit polyclonal anti phospho cdc2 tyr15  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit polyclonal anti phospho cdc2 tyr15
    Involvement of MKP-1/ERK1/2 pathway in NSC 95397-induced p21 expression and caspase-3 activation in human colon cancer. Representative Western blots showing ( A ) the expression of Cdc25A, the phosphorylation of <t>Cdk1</t> at <t>Tyr15;</t> ( B ) the expression of MKP-1 and ERK1/2, and the phosphorylation of ERK1/2 at Thr202/Tyr 204 in SW480, SW620, and DLD-1 cells treated with 10 μM NSC 95397 for 6 h, with actin as loading control; ( C ) representative Western blots showing expression of p21 and cleaved caspase-3 in colon cancer cells that were pre-incubated with 20 μM U0126 for 2 h and then treated with 10 m μM NSC 95397 for 6 h.
    Rabbit 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
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    Images

    1) Product Images from "NSC 95397 Suppresses Proliferation and Induces Apoptosis in Colon Cancer Cells through MKP-1 and the ERK1/2 Pathway"

    Article Title: NSC 95397 Suppresses Proliferation and Induces Apoptosis in Colon Cancer Cells through MKP-1 and the ERK1/2 Pathway

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19061625

    Involvement of MKP-1/ERK1/2 pathway in NSC 95397-induced p21 expression and caspase-3 activation in human colon cancer. Representative Western blots showing ( A ) the expression of Cdc25A, the phosphorylation of Cdk1 at Tyr15; ( B ) the expression of MKP-1 and ERK1/2, and the phosphorylation of ERK1/2 at Thr202/Tyr 204 in SW480, SW620, and DLD-1 cells treated with 10 μM NSC 95397 for 6 h, with actin as loading control; ( C ) representative Western blots showing expression of p21 and cleaved caspase-3 in colon cancer cells that were pre-incubated with 20 μM U0126 for 2 h and then treated with 10 m μM NSC 95397 for 6 h.
    Figure Legend Snippet: Involvement of MKP-1/ERK1/2 pathway in NSC 95397-induced p21 expression and caspase-3 activation in human colon cancer. Representative Western blots showing ( A ) the expression of Cdc25A, the phosphorylation of Cdk1 at Tyr15; ( B ) the expression of MKP-1 and ERK1/2, and the phosphorylation of ERK1/2 at Thr202/Tyr 204 in SW480, SW620, and DLD-1 cells treated with 10 μM NSC 95397 for 6 h, with actin as loading control; ( C ) representative Western blots showing expression of p21 and cleaved caspase-3 in colon cancer cells that were pre-incubated with 20 μM U0126 for 2 h and then treated with 10 m μM NSC 95397 for 6 h.

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

    rabbit polyclonal anti phospho cdk1 thr161  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit polyclonal anti phospho cdk1 thr161
    a Scheme of the experimental protocol. b TTC-stained rostral and caudal brain sections from vehicle-treated Ctrl and <t>Cdk1-cKO</t> mice and R-roscovitine-treated Ctrl mice after 1-h MCAO and 24 h of reperfusion. Scale bar = 1 mm. c Rostral and caudal quantification of infarct size (% brain area) (data represent means ± SEM; n = 6-10; *p<0.05; **p<0.005; *** p < 0.001; ANOVA with Bonferroni’s post hoc). d Quantification of neuroscore scale (data represents means ± SEM; n = 6–10; * p < 0.01; ANOVA with Bonferroni’s post hoc)
    Rabbit Polyclonal Anti Phospho Cdk1 Thr161, 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 "Genetic and pharmacological inhibition of Cdk1 provides neuroprotection towards ischemic neuronal death"

    Article Title: Genetic and pharmacological inhibition of Cdk1 provides neuroprotection towards ischemic neuronal death

    Journal: Cell Death Discovery

    doi: 10.1038/s41420-018-0044-7

    a Scheme of the experimental protocol. b TTC-stained rostral and caudal brain sections from vehicle-treated Ctrl and Cdk1-cKO mice and R-roscovitine-treated Ctrl mice after 1-h MCAO and 24 h of reperfusion. Scale bar = 1 mm. c Rostral and caudal quantification of infarct size (% brain area) (data represent means ± SEM; n = 6-10; *p<0.05; **p<0.005; *** p < 0.001; ANOVA with Bonferroni’s post hoc). d Quantification of neuroscore scale (data represents means ± SEM; n = 6–10; * p < 0.01; ANOVA with Bonferroni’s post hoc)
    Figure Legend Snippet: a Scheme of the experimental protocol. b TTC-stained rostral and caudal brain sections from vehicle-treated Ctrl and Cdk1-cKO mice and R-roscovitine-treated Ctrl mice after 1-h MCAO and 24 h of reperfusion. Scale bar = 1 mm. c Rostral and caudal quantification of infarct size (% brain area) (data represent means ± SEM; n = 6-10; *p<0.05; **p<0.005; *** p < 0.001; ANOVA with Bonferroni’s post hoc). d Quantification of neuroscore scale (data represents means ± SEM; n = 6–10; * p < 0.01; ANOVA with Bonferroni’s post hoc)

    Techniques Used: Staining

    a Scheme of the experimental protocol. b Representative confocal microscopy images and quantitative results showing no differences in the number of CC3-positive cells between normoxic and OGD condition 4 h following OGD, scale bar = 30 µm and field size = 120 µm × 70 µm (data represent means ± SEM; n = 4; ns; Student’s t -test). c Representative confocal microscopy images and quantitative results illustrating significant differences in the number of CC3-positive cells (data represent means ± SEM; n = 4; *** p < 0.001; Student’s t -test) and survival (MTT assay) (data represent means ± SEM; n = 5; * p < 0.01; Student’s t -test) between normoxic and OGD condition at 24 h following OGD. d Representative confocal images showing OGD-induced Cdk1 expression 4 h following OGD compared with normoxic condition, scale bar = 30 µm. Quantitative results represent the percentage of living (CC3-negative) neurons expressing Cdk1, field size = 120 µm × 70 µm (data represent means ± SEM; n = 5; *** p < 0.001; Student’s t -test). Western blot analysis demonstrated increased Cdk1 expression 4 h following OGD
    Figure Legend Snippet: a Scheme of the experimental protocol. b Representative confocal microscopy images and quantitative results showing no differences in the number of CC3-positive cells between normoxic and OGD condition 4 h following OGD, scale bar = 30 µm and field size = 120 µm × 70 µm (data represent means ± SEM; n = 4; ns; Student’s t -test). c Representative confocal microscopy images and quantitative results illustrating significant differences in the number of CC3-positive cells (data represent means ± SEM; n = 4; *** p < 0.001; Student’s t -test) and survival (MTT assay) (data represent means ± SEM; n = 5; * p < 0.01; Student’s t -test) between normoxic and OGD condition at 24 h following OGD. d Representative confocal images showing OGD-induced Cdk1 expression 4 h following OGD compared with normoxic condition, scale bar = 30 µm. Quantitative results represent the percentage of living (CC3-negative) neurons expressing Cdk1, field size = 120 µm × 70 µm (data represent means ± SEM; n = 5; *** p < 0.001; Student’s t -test). Western blot analysis demonstrated increased Cdk1 expression 4 h following OGD

    Techniques Used: Confocal Microscopy, MTT Assay, Expressing, Western Blot

    a Representative confocal images illustrating OGD-induced Cdk1 neuronal expression, scale bar = 30 µm. Quantitative results represent the percentage of Tuj1-positive living neurons (green) expressing Cdk1 (red), field size = 140 µm × 90 µm (data represent means ± SEM; n = 4; *** p < 0.001; ANOVA with Bonferroni’s post hoc). Western blot analysis indicated increased Cdk1 expression 1, 2 and 4 h following OGD. b Representative confocal images illustrating the number of Tuj1-positive (green) and CC3-positive (red) cells in the different conditions/genotypes 24 h following OGD, scale bar = 30 µm. Quantitative results for neuronal survival represents the number of Tuj1-positive/CC3-negative cells, field size = 230 µm × 150 µm (data represent means ± SEM; n = 5; *** p < 0.001; one-way ANOVA with Bonferroni’s post hoc). MTT assay confirm significant difference regarding neuronal survival between the different conditions 24 h following OGD (data represent means ± SEM; n = 5; *p<0.05; **p<0.01 ; one-way ANOVA with Bonferroni’s post hoc). Quantitative results for neuronal death represent the number of CC3-positive cells, field size = 230 µm × 150 µm (data represent means ± SEM; n = 4; *p<0.05; ** p < 0.005; one-way ANOVA). Western blot analysis showed decreased C-Parp (89 kDa) protein expression in Cdk1-cKO OGD-treated cultures as compared with Ctrl OGD-treated cultures
    Figure Legend Snippet: a Representative confocal images illustrating OGD-induced Cdk1 neuronal expression, scale bar = 30 µm. Quantitative results represent the percentage of Tuj1-positive living neurons (green) expressing Cdk1 (red), field size = 140 µm × 90 µm (data represent means ± SEM; n = 4; *** p < 0.001; ANOVA with Bonferroni’s post hoc). Western blot analysis indicated increased Cdk1 expression 1, 2 and 4 h following OGD. b Representative confocal images illustrating the number of Tuj1-positive (green) and CC3-positive (red) cells in the different conditions/genotypes 24 h following OGD, scale bar = 30 µm. Quantitative results for neuronal survival represents the number of Tuj1-positive/CC3-negative cells, field size = 230 µm × 150 µm (data represent means ± SEM; n = 5; *** p < 0.001; one-way ANOVA with Bonferroni’s post hoc). MTT assay confirm significant difference regarding neuronal survival between the different conditions 24 h following OGD (data represent means ± SEM; n = 5; *p<0.05; **p<0.01 ; one-way ANOVA with Bonferroni’s post hoc). Quantitative results for neuronal death represent the number of CC3-positive cells, field size = 230 µm × 150 µm (data represent means ± SEM; n = 4; *p<0.05; ** p < 0.005; one-way ANOVA). Western blot analysis showed decreased C-Parp (89 kDa) protein expression in Cdk1-cKO OGD-treated cultures as compared with Ctrl OGD-treated cultures

    Techniques Used: Expressing, Western Blot, MTT Assay

    a Photography of 2 mm thick TTC-stained mouse brain coronal section 24 h after a transient 1 h MCAO, showing the healthy cortex (red), ischemic core (white) and peri-infarct area (between red and white) in the ipsilateral cortex. b Immunohistochemistry images showing NeuN staining in the healthy part of the ipsilateral cortex and the absence of NeuN staining in the ischemic core. In between, the peri-infarct area shows re-expression of Cdk1 and P-Cdk1. Scale bar = 100 µm
    Figure Legend Snippet: a Photography of 2 mm thick TTC-stained mouse brain coronal section 24 h after a transient 1 h MCAO, showing the healthy cortex (red), ischemic core (white) and peri-infarct area (between red and white) in the ipsilateral cortex. b Immunohistochemistry images showing NeuN staining in the healthy part of the ipsilateral cortex and the absence of NeuN staining in the ischemic core. In between, the peri-infarct area shows re-expression of Cdk1 and P-Cdk1. Scale bar = 100 µm

    Techniques Used: Staining, Immunohistochemistry, Expressing

    rabbit polyclonal antibodies phospho cdc2 tyr15 antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit polyclonal antibodies phospho cdc2 tyr15 antibody
    Rabbit Polyclonal Antibodies Phospho Cdc2 Tyr15 Antibody, 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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    rabbit polyclonal phospho cdc2 tyr15 antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit polyclonal phospho cdc2 tyr15 antibody
    Cells were transfected with 500ng p16L2h or empty vector pA3M. 18h after transfection, cells were collected and stained with Propidium Iodide (PI). DNA contents were measured with Accuri ® C6 flow cytometer. A. Representative results of DNA content detection after empty vector pA3M or p16L2h plasmid transfection. B. Average percentage of the cell population in each cell cycle phase. Data are represented as mean±STD from four independent experiments. Significances of difference were analyzed with student’s t-test. *: G1: p = 0.0189; S: p = 0.0268. C. Representative Western Blotting of 500ng plasmid transfection. Average relative fold numbers shown above. Experiments were repeated three times. pRb and <t>pCdc2</t> indicate phosphorylated Rb and <t>Cdc2,</t> respectively. Protein levels were first normalized to internal control Actin and then calculated as the fold number of pA3M transfected ones. D. Two-step real-time RT-PCR of Rb and Cdc2 mRNA levels. Depicted are Ct (Cycle number of threshold) values. Results were compared to the empty vector pA3M transfected group. Data are represented as mean±STD from three independent experiments. Black bars: pA3M transfected cells; gray bars: p16L2h transfected cells. *: p = 0.012; student’s t -test.
    Rabbit Polyclonal Phospho Cdc2 Tyr15 Antibody, 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 "Host cell transcriptome modification upon exogenous HPV16 L2 protein expression"

    Article Title: Host cell transcriptome modification upon exogenous HPV16 L2 protein expression

    Journal: Oncotarget

    doi: 10.18632/oncotarget.21817

    Cells were transfected with 500ng p16L2h or empty vector pA3M. 18h after transfection, cells were collected and stained with Propidium Iodide (PI). DNA contents were measured with Accuri ® C6 flow cytometer. A. Representative results of DNA content detection after empty vector pA3M or p16L2h plasmid transfection. B. Average percentage of the cell population in each cell cycle phase. Data are represented as mean±STD from four independent experiments. Significances of difference were analyzed with student’s t-test. *: G1: p = 0.0189; S: p = 0.0268. C. Representative Western Blotting of 500ng plasmid transfection. Average relative fold numbers shown above. Experiments were repeated three times. pRb and pCdc2 indicate phosphorylated Rb and Cdc2, respectively. Protein levels were first normalized to internal control Actin and then calculated as the fold number of pA3M transfected ones. D. Two-step real-time RT-PCR of Rb and Cdc2 mRNA levels. Depicted are Ct (Cycle number of threshold) values. Results were compared to the empty vector pA3M transfected group. Data are represented as mean±STD from three independent experiments. Black bars: pA3M transfected cells; gray bars: p16L2h transfected cells. *: p = 0.012; student’s t -test.
    Figure Legend Snippet: Cells were transfected with 500ng p16L2h or empty vector pA3M. 18h after transfection, cells were collected and stained with Propidium Iodide (PI). DNA contents were measured with Accuri ® C6 flow cytometer. A. Representative results of DNA content detection after empty vector pA3M or p16L2h plasmid transfection. B. Average percentage of the cell population in each cell cycle phase. Data are represented as mean±STD from four independent experiments. Significances of difference were analyzed with student’s t-test. *: G1: p = 0.0189; S: p = 0.0268. C. Representative Western Blotting of 500ng plasmid transfection. Average relative fold numbers shown above. Experiments were repeated three times. pRb and pCdc2 indicate phosphorylated Rb and Cdc2, respectively. Protein levels were first normalized to internal control Actin and then calculated as the fold number of pA3M transfected ones. D. Two-step real-time RT-PCR of Rb and Cdc2 mRNA levels. Depicted are Ct (Cycle number of threshold) values. Results were compared to the empty vector pA3M transfected group. Data are represented as mean±STD from three independent experiments. Black bars: pA3M transfected cells; gray bars: p16L2h transfected cells. *: p = 0.012; student’s t -test.

    Techniques Used: Transfection, Plasmid Preparation, Staining, Flow Cytometry, Western Blot, Quantitative RT-PCR

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    • 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).
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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 <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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    rabbit polyclonal anti phospho cdc2 tyr15  (Cell Signaling Technology Inc)
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    (A-D) Two hours after release from a double thymidine block (DTB), synchronized HeLa S3 cells were treated with MeOH or the indicated concentrations of Pla-B. Black triangles indicate the time points of cell harvest and sample preparation (A). Cell cycle was analyzed at the indicated time points by cytometry (B). Morphology of the cells was observed under a microscope and round cells were counted at the indicated time points (C). Protein samples were prepared at the indicated time points. The protein levels of indicated proteins and phosphorylation status of <t>Cdk1</t> were analysed by immunoblotting. Protein levels of α-tubulin were analysed as an internal control (D). Error bars indicate s.d. (n = 3).
    Rabbit 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
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    rabbit polyclonal phospho cdc2 tyr15  (Cell Signaling Technology Inc)
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    A DSG (red) and the KKR (violet) motifs, and the triple K36A/K38A/R40A (KKR/AAA) Xenopus Arpp19 alanine mutant. Table depicting dephosphorylation time, B55-Arpp19 interaction and the capacity to promote mitotic entry in Arpp19-depleted extracts. B 50 ng of wild type or KKR/AAA Arpp19 mutant phosphorylated ‘in vitro’ by GwlK72M was supplemented to kinase-inactivated extracts depleted or not of the B55 protein. Arpp19 levels and of S67/S71 phosphorylation were analysed by western blotting and autoradiography, respectively. The 33 P-Arpp19/western blotting Arpp19 signal ratios were calculated using ImageJ for each time point. The percentage of the ratio remaining at each time point with respect to the ratio at 0 min was calculated and represented in bar graphs as the mean percentage ± SD; n = 3 biological independent samples. C A His-Arpp19 pulldown equivalent to 20 ng of wild type or KKR/AAA mutant was submitted to western blotting, and the amount of B55 and the levels of Arpp19 bound to the beads shown. B55/Arpp19 signal ratios were calculated using ImageJ and represented in a bar graph as the mean ratio ± SD; n = 5 biological independent samples. D Arpp19-depleted extracts were supplemented with human GwlK72M and a wild type or a KKR/AAA Arpp19 mutant and phosphorylation of Human Gwl, of <t>Tyr15,</t> of <t>Cdk1</t> and Arpp19 ectopic levels (His-Arpp19) were assessed. E A schematic of DSG regions indicating residues mutated into alanine or threonine. Table representing results on the S67/S71 dephosphorylation time and the capacities to bind B55 or to restore mitotic entry in Arpp19-depleted egg extracts of each Arpp19 form. F Wild-type Arpp19 and the indicated mutants of the DSG motif were ‘in vitro’ phosphorylated by hGwlK72M and 1 µl sample removed at 10 and 40 min, to measure S67/S71 phosphorylation by autoradiography ( P33 Arp). The amount of Arpp19 was assessed by Coomassie blue staining (Arp) 33 . P-Arpp19 levels were normalized by Arpp19 amount using Coomassie blue signal and the increase in phosphorylation between time 10 and 40 min calculated. Data from three different experiments was then used to obtain the mean ± SD and represented in a bar graph; n = 3 biological independent samples.
    Rabbit Polyclonal 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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    anti human phospho sgk rabbit polyclonal antibody  (Cell Signaling Technology Inc)
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    A DSG (red) and the KKR (violet) motifs, and the triple K36A/K38A/R40A (KKR/AAA) Xenopus Arpp19 alanine mutant. Table depicting dephosphorylation time, B55-Arpp19 interaction and the capacity to promote mitotic entry in Arpp19-depleted extracts. B 50 ng of wild type or KKR/AAA Arpp19 mutant phosphorylated ‘in vitro’ by GwlK72M was supplemented to kinase-inactivated extracts depleted or not of the B55 protein. Arpp19 levels and of S67/S71 phosphorylation were analysed by western blotting and autoradiography, respectively. The 33 P-Arpp19/western blotting Arpp19 signal ratios were calculated using ImageJ for each time point. The percentage of the ratio remaining at each time point with respect to the ratio at 0 min was calculated and represented in bar graphs as the mean percentage ± SD; n = 3 biological independent samples. C A His-Arpp19 pulldown equivalent to 20 ng of wild type or KKR/AAA mutant was submitted to western blotting, and the amount of B55 and the levels of Arpp19 bound to the beads shown. B55/Arpp19 signal ratios were calculated using ImageJ and represented in a bar graph as the mean ratio ± SD; n = 5 biological independent samples. D Arpp19-depleted extracts were supplemented with human GwlK72M and a wild type or a KKR/AAA Arpp19 mutant and phosphorylation of Human Gwl, of <t>Tyr15,</t> of <t>Cdk1</t> and Arpp19 ectopic levels (His-Arpp19) were assessed. E A schematic of DSG regions indicating residues mutated into alanine or threonine. Table representing results on the S67/S71 dephosphorylation time and the capacities to bind B55 or to restore mitotic entry in Arpp19-depleted egg extracts of each Arpp19 form. F Wild-type Arpp19 and the indicated mutants of the DSG motif were ‘in vitro’ phosphorylated by hGwlK72M and 1 µl sample removed at 10 and 40 min, to measure S67/S71 phosphorylation by autoradiography ( P33 Arp). The amount of Arpp19 was assessed by Coomassie blue staining (Arp) 33 . P-Arpp19 levels were normalized by Arpp19 amount using Coomassie blue signal and the increase in phosphorylation between time 10 and 40 min calculated. Data from three different experiments was then used to obtain the mean ± SD and represented in a bar graph; n = 3 biological independent samples.
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    rabbit polyclonal anti phospho cdk1 thr161  (Cell Signaling Technology Inc)
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    a Scheme of the experimental protocol. b TTC-stained rostral and caudal brain sections from vehicle-treated Ctrl and <t>Cdk1-cKO</t> mice and R-roscovitine-treated Ctrl mice after 1-h MCAO and 24 h of reperfusion. Scale bar = 1 mm. c Rostral and caudal quantification of infarct size (% brain area) (data represent means ± SEM; n = 6-10; *p<0.05; **p<0.005; *** p < 0.001; ANOVA with Bonferroni’s post hoc). d Quantification of neuroscore scale (data represents means ± SEM; n = 6–10; * p < 0.01; ANOVA with Bonferroni’s post hoc)
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    rabbit polyclonal antibodies phospho cdc2 tyr15 antibody  (Cell Signaling Technology Inc)
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    a Scheme of the experimental protocol. b TTC-stained rostral and caudal brain sections from vehicle-treated Ctrl and <t>Cdk1-cKO</t> mice and R-roscovitine-treated Ctrl mice after 1-h MCAO and 24 h of reperfusion. Scale bar = 1 mm. c Rostral and caudal quantification of infarct size (% brain area) (data represent means ± SEM; n = 6-10; *p<0.05; **p<0.005; *** p < 0.001; ANOVA with Bonferroni’s post hoc). d Quantification of neuroscore scale (data represents means ± SEM; n = 6–10; * p < 0.01; ANOVA with Bonferroni’s post hoc)
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    Cells were transfected with 500ng p16L2h or empty vector pA3M. 18h after transfection, cells were collected and stained with Propidium Iodide (PI). DNA contents were measured with Accuri ® C6 flow cytometer. A. Representative results of DNA content detection after empty vector pA3M or p16L2h plasmid transfection. B. Average percentage of the cell population in each cell cycle phase. Data are represented as mean±STD from four independent experiments. Significances of difference were analyzed with student’s t-test. *: G1: p = 0.0189; S: p = 0.0268. C. Representative Western Blotting of 500ng plasmid transfection. Average relative fold numbers shown above. Experiments were repeated three times. pRb and <t>pCdc2</t> indicate phosphorylated Rb and <t>Cdc2,</t> respectively. Protein levels were first normalized to internal control Actin and then calculated as the fold number of pA3M transfected ones. D. Two-step real-time RT-PCR of Rb and Cdc2 mRNA levels. Depicted are Ct (Cycle number of threshold) values. Results were compared to the empty vector pA3M transfected group. Data are represented as mean±STD from three independent experiments. Black bars: pA3M transfected cells; gray bars: p16L2h transfected cells. *: p = 0.012; student’s t -test.
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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-D) Two hours after release from a double thymidine block (DTB), synchronized HeLa S3 cells were treated with MeOH or the indicated concentrations of Pla-B. Black triangles indicate the time points of cell harvest and sample preparation (A). Cell cycle was analyzed at the indicated time points by cytometry (B). Morphology of the cells was observed under a microscope and round cells were counted at the indicated time points (C). Protein samples were prepared at the indicated time points. The protein levels of indicated proteins and phosphorylation status of Cdk1 were analysed by immunoblotting. Protein levels of α-tubulin were analysed as an internal control (D). Error bars indicate s.d. (n = 3).

    Journal: bioRxiv

    Article Title: A truncated form of the p27 CDK inhibitor translated from pre-mRNA causes cell cycle arrest at G2 phase

    doi: 10.1101/2022.01.12.476115

    Figure Lengend Snippet: (A-D) Two hours after release from a double thymidine block (DTB), synchronized HeLa S3 cells were treated with MeOH or the indicated concentrations of Pla-B. Black triangles indicate the time points of cell harvest and sample preparation (A). Cell cycle was analyzed at the indicated time points by cytometry (B). Morphology of the cells was observed under a microscope and round cells were counted at the indicated time points (C). Protein samples were prepared at the indicated time points. The protein levels of indicated proteins and phosphorylation status of Cdk1 were analysed by immunoblotting. Protein levels of α-tubulin were analysed as an internal control (D). Error bars indicate s.d. (n = 3).

    Article Snippet: Mouse monoclonal anti-Cyclin A2 (#4656), rabbit polyclonal anti-Cyclin B1 (#4138), mouse monoclonal anti-cdc2 (#9116), rabbit polyclonal anti-phospho-cdc2 (Tyr15) (#9111), mouse monoclonal anti-Cyclin E1 (#4129), rabbit monoclonal anti-Plk2 (#14812), rabbit monoclonal anti-Wee1 (#13084) and rabbit monoclonal anti-p27 (#3686) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Blocking Assay, Sample Prep, Cytometry, Microscopy, Western Blot

    (A) Cells expressing Flag-p27* under the control of tetracycline were synchronized by a double thymidine block. The cells were treated with 1 μg/ml DOX at the same time as release from the double thymidine block. The cells were harvested at 8 h after release from the double thymidine block (G2/M phase) and then immunoprecipitation was performed using anti-DDDDK (Flag) antibodies. Flag-tagged and coimmunoprecipitated proteins were analysed by immunoblotting. (B, C) Purified Flag-tagged proteins were applied to an in vitro kinase assay reaction and kinase activities of Cyclin A2/Cdk1 (B) and Cyclin B1/Cdk1 (C) were measured. Statistical significance was assessed by the one-way ANOVA and Dunnett’s test (*: P < 0.05; **: P < 0.01; ***: P < 0.01). Error bars indicate s.d. (n = 3).

    Journal: bioRxiv

    Article Title: A truncated form of the p27 CDK inhibitor translated from pre-mRNA causes cell cycle arrest at G2 phase

    doi: 10.1101/2022.01.12.476115

    Figure Lengend Snippet: (A) Cells expressing Flag-p27* under the control of tetracycline were synchronized by a double thymidine block. The cells were treated with 1 μg/ml DOX at the same time as release from the double thymidine block. The cells were harvested at 8 h after release from the double thymidine block (G2/M phase) and then immunoprecipitation was performed using anti-DDDDK (Flag) antibodies. Flag-tagged and coimmunoprecipitated proteins were analysed by immunoblotting. (B, C) Purified Flag-tagged proteins were applied to an in vitro kinase assay reaction and kinase activities of Cyclin A2/Cdk1 (B) and Cyclin B1/Cdk1 (C) were measured. Statistical significance was assessed by the one-way ANOVA and Dunnett’s test (*: P < 0.05; **: P < 0.01; ***: P < 0.01). Error bars indicate s.d. (n = 3).

    Article Snippet: Mouse monoclonal anti-Cyclin A2 (#4656), rabbit polyclonal anti-Cyclin B1 (#4138), mouse monoclonal anti-cdc2 (#9116), rabbit polyclonal anti-phospho-cdc2 (Tyr15) (#9111), mouse monoclonal anti-Cyclin E1 (#4129), rabbit monoclonal anti-Plk2 (#14812), rabbit monoclonal anti-Wee1 (#13084) and rabbit monoclonal anti-p27 (#3686) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Expressing, Blocking Assay, Immunoprecipitation, Western Blot, Purification, In Vitro, Kinase Assay

    A DSG (red) and the KKR (violet) motifs, and the triple K36A/K38A/R40A (KKR/AAA) Xenopus Arpp19 alanine mutant. Table depicting dephosphorylation time, B55-Arpp19 interaction and the capacity to promote mitotic entry in Arpp19-depleted extracts. B 50 ng of wild type or KKR/AAA Arpp19 mutant phosphorylated ‘in vitro’ by GwlK72M was supplemented to kinase-inactivated extracts depleted or not of the B55 protein. Arpp19 levels and of S67/S71 phosphorylation were analysed by western blotting and autoradiography, respectively. The 33 P-Arpp19/western blotting Arpp19 signal ratios were calculated using ImageJ for each time point. The percentage of the ratio remaining at each time point with respect to the ratio at 0 min was calculated and represented in bar graphs as the mean percentage ± SD; n = 3 biological independent samples. C A His-Arpp19 pulldown equivalent to 20 ng of wild type or KKR/AAA mutant was submitted to western blotting, and the amount of B55 and the levels of Arpp19 bound to the beads shown. B55/Arpp19 signal ratios were calculated using ImageJ and represented in a bar graph as the mean ratio ± SD; n = 5 biological independent samples. D Arpp19-depleted extracts were supplemented with human GwlK72M and a wild type or a KKR/AAA Arpp19 mutant and phosphorylation of Human Gwl, of Tyr15, of Cdk1 and Arpp19 ectopic levels (His-Arpp19) were assessed. E A schematic of DSG regions indicating residues mutated into alanine or threonine. Table representing results on the S67/S71 dephosphorylation time and the capacities to bind B55 or to restore mitotic entry in Arpp19-depleted egg extracts of each Arpp19 form. F Wild-type Arpp19 and the indicated mutants of the DSG motif were ‘in vitro’ phosphorylated by hGwlK72M and 1 µl sample removed at 10 and 40 min, to measure S67/S71 phosphorylation by autoradiography ( P33 Arp). The amount of Arpp19 was assessed by Coomassie blue staining (Arp) 33 . P-Arpp19 levels were normalized by Arpp19 amount using Coomassie blue signal and the increase in phosphorylation between time 10 and 40 min calculated. Data from three different experiments was then used to obtain the mean ± SD and represented in a bar graph; n = 3 biological independent samples.

    Journal: Nature Communications

    Article Title: The study of the determinants controlling Arpp19 phosphatase-inhibitory activity reveals an Arpp19/PP2A-B55 feedback loop

    doi: 10.1038/s41467-021-23657-0

    Figure Lengend Snippet: A DSG (red) and the KKR (violet) motifs, and the triple K36A/K38A/R40A (KKR/AAA) Xenopus Arpp19 alanine mutant. Table depicting dephosphorylation time, B55-Arpp19 interaction and the capacity to promote mitotic entry in Arpp19-depleted extracts. B 50 ng of wild type or KKR/AAA Arpp19 mutant phosphorylated ‘in vitro’ by GwlK72M was supplemented to kinase-inactivated extracts depleted or not of the B55 protein. Arpp19 levels and of S67/S71 phosphorylation were analysed by western blotting and autoradiography, respectively. The 33 P-Arpp19/western blotting Arpp19 signal ratios were calculated using ImageJ for each time point. The percentage of the ratio remaining at each time point with respect to the ratio at 0 min was calculated and represented in bar graphs as the mean percentage ± SD; n = 3 biological independent samples. C A His-Arpp19 pulldown equivalent to 20 ng of wild type or KKR/AAA mutant was submitted to western blotting, and the amount of B55 and the levels of Arpp19 bound to the beads shown. B55/Arpp19 signal ratios were calculated using ImageJ and represented in a bar graph as the mean ratio ± SD; n = 5 biological independent samples. D Arpp19-depleted extracts were supplemented with human GwlK72M and a wild type or a KKR/AAA Arpp19 mutant and phosphorylation of Human Gwl, of Tyr15, of Cdk1 and Arpp19 ectopic levels (His-Arpp19) were assessed. E A schematic of DSG regions indicating residues mutated into alanine or threonine. Table representing results on the S67/S71 dephosphorylation time and the capacities to bind B55 or to restore mitotic entry in Arpp19-depleted egg extracts of each Arpp19 form. F Wild-type Arpp19 and the indicated mutants of the DSG motif were ‘in vitro’ phosphorylated by hGwlK72M and 1 µl sample removed at 10 and 40 min, to measure S67/S71 phosphorylation by autoradiography ( P33 Arp). The amount of Arpp19 was assessed by Coomassie blue staining (Arp) 33 . P-Arpp19 levels were normalized by Arpp19 amount using Coomassie blue signal and the increase in phosphorylation between time 10 and 40 min calculated. Data from three different experiments was then used to obtain the mean ± SD and represented in a bar graph; n = 3 biological independent samples.

    Article Snippet: The antibodies used in this study are the following: Rabbit Polyclonal anti-Human Gwl , Rabbit Polyclonal Phospho-Cdc2 (Tyr15) (Cell Signaling Technology Cat#9111), Rabbit Polyclonal anti- Xenopus Arpp19 , Rabbit Polyclonal anti-PP2A/B55δ (Cell Signaling Technology Cat#2290), Rabbit Polyclonal anti- Xenopus Cdc27 , Rabbit Polyclonal anti- Xenopus Cyclin B2 , Rabbit Polyclonal anti- Xenopus Cdk1 , Rabbit Monoclonal PhosphoThr320 of PP1 (Abcam Cat#62334), Mouse Monoclonal Phospho-Erk (Cell Signaling Cat# 9106 S), Rabbit Polyclonal anti-phosphorylated Arpp19 (S67) (Cell Signaling Cat#5240 S), Rabbit Polyclonal anti-PRC1 (Santa Cruz Cat# 376982), Goat Polyclonal anti-phosphorylated PRC1 (T481) (Santa Cruz Cat#11768), Mouse Monoclonal anti-PP2A (C) subunit-α (isoform Merck Millipore Cat#05-42), Rat Polyclonal PP2A (A) subunit (Cell Signaling Cat#2260), Mouse Monoclonal anti-PP1 kindly gifted by Dr M Bollen and used in Ma et al. , Anti-PP4c (Bethyl Cat#A300-835A), Anti-PP6 (Santa Cruz Cat#393294), Goat anti-rat IgG-horseradish peroxidase (HRP) (Santa Cruz Cat#2006), HRP-conjugated anti-Rabbit secondary antibodies (Cell Signalling Technology Cat#7074), Donkey anti-goat IgG-HRP (Santa Cruz Cat# sc-2020), Rabbit Polyclonal anti-phosphorylated Arpp19 (S109/S113) (this study), and Rabbit Polyclonal anti-PP2A (B56) γ-subunit (this study).

    Techniques: Mutagenesis, De-Phosphorylation Assay, In Vitro, Western Blot, Autoradiography, Staining

    A B55 levels associated to 20 ng of wild type or the indicated DSG mutants of His-Arpp19-pulldowns. Arpp19 amount in these pulldowns is also shown. Data were represented as mean B55/Arpp19 ratio ± SD. Two-tailed unpaired Student’s t -tests were performed in each pulldown to determine statistical relevance. p vs. wild-type Arpp19 is shown; n = 3 biological independent samples for mutants Y68A, G72A and Y74A; n = 5 for the D73A, n = 6 for the wild-type form and n = 4 for the rest. B The wild type and the indicated DSG mutant forms of Arpp19 were thio-phosphorylated and used for His-pulldown. B55 and Arpp19 levels were checked by western blotting and shown. The B55/Arpp19 ratios were quantified and represented in a bar graph as mean ± SD. Two-tailed unpaired Student t -tests were performed in each pulldown to determine statistical relevance. p vs. wild-type Arpp19 is shown; n = 3 biological independent samples. C The B55/Arpp19 ratios were obtained as in A for the indicated mutants and represented in a bar graph as mean ± SD. Two-tailed unpaired; p vs. wild-type Arpp19 is shown; n = 3 biological independent samples for the wild-type form; n = 8 for the G72A mutant, n = 5 for the G72A-S71A mutant and n = 3 for the G72A-S71T mutant. D Prophase oocytes were injected or not (PG) with 50 ng of the wild-type His-Arpp19 protein or with the indicated mutant forms and, 1 h later, treated with progesterone. GVBD was then scored as a function of time. Germinal vesicle (GV) and mature oocytes (GVBD) were western blotted to determine the levels of the injected protein, as well as the phosphorylation of Gwl and of the inhibitory site of Cdk1 Tyrosine 15. E As for E , except that the indicated mutant form of Arpp19 was used.

    Journal: Nature Communications

    Article Title: The study of the determinants controlling Arpp19 phosphatase-inhibitory activity reveals an Arpp19/PP2A-B55 feedback loop

    doi: 10.1038/s41467-021-23657-0

    Figure Lengend Snippet: A B55 levels associated to 20 ng of wild type or the indicated DSG mutants of His-Arpp19-pulldowns. Arpp19 amount in these pulldowns is also shown. Data were represented as mean B55/Arpp19 ratio ± SD. Two-tailed unpaired Student’s t -tests were performed in each pulldown to determine statistical relevance. p vs. wild-type Arpp19 is shown; n = 3 biological independent samples for mutants Y68A, G72A and Y74A; n = 5 for the D73A, n = 6 for the wild-type form and n = 4 for the rest. B The wild type and the indicated DSG mutant forms of Arpp19 were thio-phosphorylated and used for His-pulldown. B55 and Arpp19 levels were checked by western blotting and shown. The B55/Arpp19 ratios were quantified and represented in a bar graph as mean ± SD. Two-tailed unpaired Student t -tests were performed in each pulldown to determine statistical relevance. p vs. wild-type Arpp19 is shown; n = 3 biological independent samples. C The B55/Arpp19 ratios were obtained as in A for the indicated mutants and represented in a bar graph as mean ± SD. Two-tailed unpaired; p vs. wild-type Arpp19 is shown; n = 3 biological independent samples for the wild-type form; n = 8 for the G72A mutant, n = 5 for the G72A-S71A mutant and n = 3 for the G72A-S71T mutant. D Prophase oocytes were injected or not (PG) with 50 ng of the wild-type His-Arpp19 protein or with the indicated mutant forms and, 1 h later, treated with progesterone. GVBD was then scored as a function of time. Germinal vesicle (GV) and mature oocytes (GVBD) were western blotted to determine the levels of the injected protein, as well as the phosphorylation of Gwl and of the inhibitory site of Cdk1 Tyrosine 15. E As for E , except that the indicated mutant form of Arpp19 was used.

    Article Snippet: The antibodies used in this study are the following: Rabbit Polyclonal anti-Human Gwl , Rabbit Polyclonal Phospho-Cdc2 (Tyr15) (Cell Signaling Technology Cat#9111), Rabbit Polyclonal anti- Xenopus Arpp19 , Rabbit Polyclonal anti-PP2A/B55δ (Cell Signaling Technology Cat#2290), Rabbit Polyclonal anti- Xenopus Cdc27 , Rabbit Polyclonal anti- Xenopus Cyclin B2 , Rabbit Polyclonal anti- Xenopus Cdk1 , Rabbit Monoclonal PhosphoThr320 of PP1 (Abcam Cat#62334), Mouse Monoclonal Phospho-Erk (Cell Signaling Cat# 9106 S), Rabbit Polyclonal anti-phosphorylated Arpp19 (S67) (Cell Signaling Cat#5240 S), Rabbit Polyclonal anti-PRC1 (Santa Cruz Cat# 376982), Goat Polyclonal anti-phosphorylated PRC1 (T481) (Santa Cruz Cat#11768), Mouse Monoclonal anti-PP2A (C) subunit-α (isoform Merck Millipore Cat#05-42), Rat Polyclonal PP2A (A) subunit (Cell Signaling Cat#2260), Mouse Monoclonal anti-PP1 kindly gifted by Dr M Bollen and used in Ma et al. , Anti-PP4c (Bethyl Cat#A300-835A), Anti-PP6 (Santa Cruz Cat#393294), Goat anti-rat IgG-horseradish peroxidase (HRP) (Santa Cruz Cat#2006), HRP-conjugated anti-Rabbit secondary antibodies (Cell Signalling Technology Cat#7074), Donkey anti-goat IgG-HRP (Santa Cruz Cat# sc-2020), Rabbit Polyclonal anti-phosphorylated Arpp19 (S109/S113) (this study), and Rabbit Polyclonal anti-PP2A (B56) γ-subunit (this study).

    Techniques: Two Tailed Test, Mutagenesis, Western Blot, Injection

    A Wild type and S109/S113D Arpp19 mutant were phosphorylated ‘in vitro’ with of [γ 33 P] ATP by GwlK72M on S67/S71 and supplemented to kinase-inactivated Xenopus egg extracts. The dephosphorylation of this residue was analysed at the indicated times by autoradiography ( P33 Arp) and the amount of Arpp19 in each sample measured by western blotting (Arp). B CytoStatic Factor (CSF) egg extracts were supplemented with a trace level of Arpp19-purified protein and activated to exit meiosis by the addition of active CamKII. The levels and dephosphorylation of the indicated proteins were analysed by western blotting, whereas Cyclin B/Cdk1 activity was measured by histone H1 phosphorylation (H1K). ‘Inter’ denotes interphase egg extracts. C As for C , except that S67/S71 Arpp19 dephosphorylation and PP2A-B55 reactivation upon meiosis exit in these extracts was blocked by the concomitant addition of GwlK72M-purified protein.

    Journal: Nature Communications

    Article Title: The study of the determinants controlling Arpp19 phosphatase-inhibitory activity reveals an Arpp19/PP2A-B55 feedback loop

    doi: 10.1038/s41467-021-23657-0

    Figure Lengend Snippet: A Wild type and S109/S113D Arpp19 mutant were phosphorylated ‘in vitro’ with of [γ 33 P] ATP by GwlK72M on S67/S71 and supplemented to kinase-inactivated Xenopus egg extracts. The dephosphorylation of this residue was analysed at the indicated times by autoradiography ( P33 Arp) and the amount of Arpp19 in each sample measured by western blotting (Arp). B CytoStatic Factor (CSF) egg extracts were supplemented with a trace level of Arpp19-purified protein and activated to exit meiosis by the addition of active CamKII. The levels and dephosphorylation of the indicated proteins were analysed by western blotting, whereas Cyclin B/Cdk1 activity was measured by histone H1 phosphorylation (H1K). ‘Inter’ denotes interphase egg extracts. C As for C , except that S67/S71 Arpp19 dephosphorylation and PP2A-B55 reactivation upon meiosis exit in these extracts was blocked by the concomitant addition of GwlK72M-purified protein.

    Article Snippet: The antibodies used in this study are the following: Rabbit Polyclonal anti-Human Gwl , Rabbit Polyclonal Phospho-Cdc2 (Tyr15) (Cell Signaling Technology Cat#9111), Rabbit Polyclonal anti- Xenopus Arpp19 , Rabbit Polyclonal anti-PP2A/B55δ (Cell Signaling Technology Cat#2290), Rabbit Polyclonal anti- Xenopus Cdc27 , Rabbit Polyclonal anti- Xenopus Cyclin B2 , Rabbit Polyclonal anti- Xenopus Cdk1 , Rabbit Monoclonal PhosphoThr320 of PP1 (Abcam Cat#62334), Mouse Monoclonal Phospho-Erk (Cell Signaling Cat# 9106 S), Rabbit Polyclonal anti-phosphorylated Arpp19 (S67) (Cell Signaling Cat#5240 S), Rabbit Polyclonal anti-PRC1 (Santa Cruz Cat# 376982), Goat Polyclonal anti-phosphorylated PRC1 (T481) (Santa Cruz Cat#11768), Mouse Monoclonal anti-PP2A (C) subunit-α (isoform Merck Millipore Cat#05-42), Rat Polyclonal PP2A (A) subunit (Cell Signaling Cat#2260), Mouse Monoclonal anti-PP1 kindly gifted by Dr M Bollen and used in Ma et al. , Anti-PP4c (Bethyl Cat#A300-835A), Anti-PP6 (Santa Cruz Cat#393294), Goat anti-rat IgG-horseradish peroxidase (HRP) (Santa Cruz Cat#2006), HRP-conjugated anti-Rabbit secondary antibodies (Cell Signalling Technology Cat#7074), Donkey anti-goat IgG-HRP (Santa Cruz Cat# sc-2020), Rabbit Polyclonal anti-phosphorylated Arpp19 (S109/S113) (this study), and Rabbit Polyclonal anti-PP2A (B56) γ-subunit (this study).

    Techniques: Mutagenesis, In Vitro, De-Phosphorylation Assay, Autoradiography, Western Blot, Purification, Activity Assay

    a Scheme of the experimental protocol. b TTC-stained rostral and caudal brain sections from vehicle-treated Ctrl and Cdk1-cKO mice and R-roscovitine-treated Ctrl mice after 1-h MCAO and 24 h of reperfusion. Scale bar = 1 mm. c Rostral and caudal quantification of infarct size (% brain area) (data represent means ± SEM; n = 6-10; *p<0.05; **p<0.005; *** p < 0.001; ANOVA with Bonferroni’s post hoc). d Quantification of neuroscore scale (data represents means ± SEM; n = 6–10; * p < 0.01; ANOVA with Bonferroni’s post hoc)

    Journal: Cell Death Discovery

    Article Title: Genetic and pharmacological inhibition of Cdk1 provides neuroprotection towards ischemic neuronal death

    doi: 10.1038/s41420-018-0044-7

    Figure Lengend Snippet: a Scheme of the experimental protocol. b TTC-stained rostral and caudal brain sections from vehicle-treated Ctrl and Cdk1-cKO mice and R-roscovitine-treated Ctrl mice after 1-h MCAO and 24 h of reperfusion. Scale bar = 1 mm. c Rostral and caudal quantification of infarct size (% brain area) (data represent means ± SEM; n = 6-10; *p<0.05; **p<0.005; *** p < 0.001; ANOVA with Bonferroni’s post hoc). d Quantification of neuroscore scale (data represents means ± SEM; n = 6–10; * p < 0.01; ANOVA with Bonferroni’s post hoc)

    Article Snippet: The following antibodies were used at the indicated dilutions: rabbit monoclonal anti-β-III tubulin (Tuj1) (1:500; BioLegend cat# 801202, RRID:AB_10063408), rabbit polyclonal anti-phospho-Cdk1 (Thr161) (1:1000; Cell Signaling Technology cat# 9111S, RRID:AB_331460), rabbit anti-Cdk1 (1:500; Atlas Antibodies cat# HPA003387, RRID:AB_1846356), rabbit anti-CC3 (1:1000; Cell Signaling Technology cat# 9661, RRID:AB_2341188), mouse anti-GFAP (1:500; Sigma-Aldrich cat# G3893, RRID:AB_477010), mouse monoclonal anti-Cdk1 (1:500; BD Biosciences cat# 612306, RRID:AB_399621), mouse anti-NeuN (1:500, Millipore cat# MAB377, RRID:AB_2298772).

    Techniques: Staining

    a Scheme of the experimental protocol. b Representative confocal microscopy images and quantitative results showing no differences in the number of CC3-positive cells between normoxic and OGD condition 4 h following OGD, scale bar = 30 µm and field size = 120 µm × 70 µm (data represent means ± SEM; n = 4; ns; Student’s t -test). c Representative confocal microscopy images and quantitative results illustrating significant differences in the number of CC3-positive cells (data represent means ± SEM; n = 4; *** p < 0.001; Student’s t -test) and survival (MTT assay) (data represent means ± SEM; n = 5; * p < 0.01; Student’s t -test) between normoxic and OGD condition at 24 h following OGD. d Representative confocal images showing OGD-induced Cdk1 expression 4 h following OGD compared with normoxic condition, scale bar = 30 µm. Quantitative results represent the percentage of living (CC3-negative) neurons expressing Cdk1, field size = 120 µm × 70 µm (data represent means ± SEM; n = 5; *** p < 0.001; Student’s t -test). Western blot analysis demonstrated increased Cdk1 expression 4 h following OGD

    Journal: Cell Death Discovery

    Article Title: Genetic and pharmacological inhibition of Cdk1 provides neuroprotection towards ischemic neuronal death

    doi: 10.1038/s41420-018-0044-7

    Figure Lengend Snippet: a Scheme of the experimental protocol. b Representative confocal microscopy images and quantitative results showing no differences in the number of CC3-positive cells between normoxic and OGD condition 4 h following OGD, scale bar = 30 µm and field size = 120 µm × 70 µm (data represent means ± SEM; n = 4; ns; Student’s t -test). c Representative confocal microscopy images and quantitative results illustrating significant differences in the number of CC3-positive cells (data represent means ± SEM; n = 4; *** p < 0.001; Student’s t -test) and survival (MTT assay) (data represent means ± SEM; n = 5; * p < 0.01; Student’s t -test) between normoxic and OGD condition at 24 h following OGD. d Representative confocal images showing OGD-induced Cdk1 expression 4 h following OGD compared with normoxic condition, scale bar = 30 µm. Quantitative results represent the percentage of living (CC3-negative) neurons expressing Cdk1, field size = 120 µm × 70 µm (data represent means ± SEM; n = 5; *** p < 0.001; Student’s t -test). Western blot analysis demonstrated increased Cdk1 expression 4 h following OGD

    Article Snippet: The following antibodies were used at the indicated dilutions: rabbit monoclonal anti-β-III tubulin (Tuj1) (1:500; BioLegend cat# 801202, RRID:AB_10063408), rabbit polyclonal anti-phospho-Cdk1 (Thr161) (1:1000; Cell Signaling Technology cat# 9111S, RRID:AB_331460), rabbit anti-Cdk1 (1:500; Atlas Antibodies cat# HPA003387, RRID:AB_1846356), rabbit anti-CC3 (1:1000; Cell Signaling Technology cat# 9661, RRID:AB_2341188), mouse anti-GFAP (1:500; Sigma-Aldrich cat# G3893, RRID:AB_477010), mouse monoclonal anti-Cdk1 (1:500; BD Biosciences cat# 612306, RRID:AB_399621), mouse anti-NeuN (1:500, Millipore cat# MAB377, RRID:AB_2298772).

    Techniques: Confocal Microscopy, MTT Assay, Expressing, Western Blot

    a Representative confocal images illustrating OGD-induced Cdk1 neuronal expression, scale bar = 30 µm. Quantitative results represent the percentage of Tuj1-positive living neurons (green) expressing Cdk1 (red), field size = 140 µm × 90 µm (data represent means ± SEM; n = 4; *** p < 0.001; ANOVA with Bonferroni’s post hoc). Western blot analysis indicated increased Cdk1 expression 1, 2 and 4 h following OGD. b Representative confocal images illustrating the number of Tuj1-positive (green) and CC3-positive (red) cells in the different conditions/genotypes 24 h following OGD, scale bar = 30 µm. Quantitative results for neuronal survival represents the number of Tuj1-positive/CC3-negative cells, field size = 230 µm × 150 µm (data represent means ± SEM; n = 5; *** p < 0.001; one-way ANOVA with Bonferroni’s post hoc). MTT assay confirm significant difference regarding neuronal survival between the different conditions 24 h following OGD (data represent means ± SEM; n = 5; *p<0.05; **p<0.01 ; one-way ANOVA with Bonferroni’s post hoc). Quantitative results for neuronal death represent the number of CC3-positive cells, field size = 230 µm × 150 µm (data represent means ± SEM; n = 4; *p<0.05; ** p < 0.005; one-way ANOVA). Western blot analysis showed decreased C-Parp (89 kDa) protein expression in Cdk1-cKO OGD-treated cultures as compared with Ctrl OGD-treated cultures

    Journal: Cell Death Discovery

    Article Title: Genetic and pharmacological inhibition of Cdk1 provides neuroprotection towards ischemic neuronal death

    doi: 10.1038/s41420-018-0044-7

    Figure Lengend Snippet: a Representative confocal images illustrating OGD-induced Cdk1 neuronal expression, scale bar = 30 µm. Quantitative results represent the percentage of Tuj1-positive living neurons (green) expressing Cdk1 (red), field size = 140 µm × 90 µm (data represent means ± SEM; n = 4; *** p < 0.001; ANOVA with Bonferroni’s post hoc). Western blot analysis indicated increased Cdk1 expression 1, 2 and 4 h following OGD. b Representative confocal images illustrating the number of Tuj1-positive (green) and CC3-positive (red) cells in the different conditions/genotypes 24 h following OGD, scale bar = 30 µm. Quantitative results for neuronal survival represents the number of Tuj1-positive/CC3-negative cells, field size = 230 µm × 150 µm (data represent means ± SEM; n = 5; *** p < 0.001; one-way ANOVA with Bonferroni’s post hoc). MTT assay confirm significant difference regarding neuronal survival between the different conditions 24 h following OGD (data represent means ± SEM; n = 5; *p<0.05; **p<0.01 ; one-way ANOVA with Bonferroni’s post hoc). Quantitative results for neuronal death represent the number of CC3-positive cells, field size = 230 µm × 150 µm (data represent means ± SEM; n = 4; *p<0.05; ** p < 0.005; one-way ANOVA). Western blot analysis showed decreased C-Parp (89 kDa) protein expression in Cdk1-cKO OGD-treated cultures as compared with Ctrl OGD-treated cultures

    Article Snippet: The following antibodies were used at the indicated dilutions: rabbit monoclonal anti-β-III tubulin (Tuj1) (1:500; BioLegend cat# 801202, RRID:AB_10063408), rabbit polyclonal anti-phospho-Cdk1 (Thr161) (1:1000; Cell Signaling Technology cat# 9111S, RRID:AB_331460), rabbit anti-Cdk1 (1:500; Atlas Antibodies cat# HPA003387, RRID:AB_1846356), rabbit anti-CC3 (1:1000; Cell Signaling Technology cat# 9661, RRID:AB_2341188), mouse anti-GFAP (1:500; Sigma-Aldrich cat# G3893, RRID:AB_477010), mouse monoclonal anti-Cdk1 (1:500; BD Biosciences cat# 612306, RRID:AB_399621), mouse anti-NeuN (1:500, Millipore cat# MAB377, RRID:AB_2298772).

    Techniques: Expressing, Western Blot, MTT Assay

    a Photography of 2 mm thick TTC-stained mouse brain coronal section 24 h after a transient 1 h MCAO, showing the healthy cortex (red), ischemic core (white) and peri-infarct area (between red and white) in the ipsilateral cortex. b Immunohistochemistry images showing NeuN staining in the healthy part of the ipsilateral cortex and the absence of NeuN staining in the ischemic core. In between, the peri-infarct area shows re-expression of Cdk1 and P-Cdk1. Scale bar = 100 µm

    Journal: Cell Death Discovery

    Article Title: Genetic and pharmacological inhibition of Cdk1 provides neuroprotection towards ischemic neuronal death

    doi: 10.1038/s41420-018-0044-7

    Figure Lengend Snippet: a Photography of 2 mm thick TTC-stained mouse brain coronal section 24 h after a transient 1 h MCAO, showing the healthy cortex (red), ischemic core (white) and peri-infarct area (between red and white) in the ipsilateral cortex. b Immunohistochemistry images showing NeuN staining in the healthy part of the ipsilateral cortex and the absence of NeuN staining in the ischemic core. In between, the peri-infarct area shows re-expression of Cdk1 and P-Cdk1. Scale bar = 100 µm

    Article Snippet: The following antibodies were used at the indicated dilutions: rabbit monoclonal anti-β-III tubulin (Tuj1) (1:500; BioLegend cat# 801202, RRID:AB_10063408), rabbit polyclonal anti-phospho-Cdk1 (Thr161) (1:1000; Cell Signaling Technology cat# 9111S, RRID:AB_331460), rabbit anti-Cdk1 (1:500; Atlas Antibodies cat# HPA003387, RRID:AB_1846356), rabbit anti-CC3 (1:1000; Cell Signaling Technology cat# 9661, RRID:AB_2341188), mouse anti-GFAP (1:500; Sigma-Aldrich cat# G3893, RRID:AB_477010), mouse monoclonal anti-Cdk1 (1:500; BD Biosciences cat# 612306, RRID:AB_399621), mouse anti-NeuN (1:500, Millipore cat# MAB377, RRID:AB_2298772).

    Techniques: Staining, Immunohistochemistry, Expressing

    Cells were transfected with 500ng p16L2h or empty vector pA3M. 18h after transfection, cells were collected and stained with Propidium Iodide (PI). DNA contents were measured with Accuri ® C6 flow cytometer. A. Representative results of DNA content detection after empty vector pA3M or p16L2h plasmid transfection. B. Average percentage of the cell population in each cell cycle phase. Data are represented as mean±STD from four independent experiments. Significances of difference were analyzed with student’s t-test. *: G1: p = 0.0189; S: p = 0.0268. C. Representative Western Blotting of 500ng plasmid transfection. Average relative fold numbers shown above. Experiments were repeated three times. pRb and pCdc2 indicate phosphorylated Rb and Cdc2, respectively. Protein levels were first normalized to internal control Actin and then calculated as the fold number of pA3M transfected ones. D. Two-step real-time RT-PCR of Rb and Cdc2 mRNA levels. Depicted are Ct (Cycle number of threshold) values. Results were compared to the empty vector pA3M transfected group. Data are represented as mean±STD from three independent experiments. Black bars: pA3M transfected cells; gray bars: p16L2h transfected cells. *: p = 0.012; student’s t -test.

    Journal: Oncotarget

    Article Title: Host cell transcriptome modification upon exogenous HPV16 L2 protein expression

    doi: 10.18632/oncotarget.21817

    Figure Lengend Snippet: Cells were transfected with 500ng p16L2h or empty vector pA3M. 18h after transfection, cells were collected and stained with Propidium Iodide (PI). DNA contents were measured with Accuri ® C6 flow cytometer. A. Representative results of DNA content detection after empty vector pA3M or p16L2h plasmid transfection. B. Average percentage of the cell population in each cell cycle phase. Data are represented as mean±STD from four independent experiments. Significances of difference were analyzed with student’s t-test. *: G1: p = 0.0189; S: p = 0.0268. C. Representative Western Blotting of 500ng plasmid transfection. Average relative fold numbers shown above. Experiments were repeated three times. pRb and pCdc2 indicate phosphorylated Rb and Cdc2, respectively. Protein levels were first normalized to internal control Actin and then calculated as the fold number of pA3M transfected ones. D. Two-step real-time RT-PCR of Rb and Cdc2 mRNA levels. Depicted are Ct (Cycle number of threshold) values. Results were compared to the empty vector pA3M transfected group. Data are represented as mean±STD from three independent experiments. Black bars: pA3M transfected cells; gray bars: p16L2h transfected cells. *: p = 0.012; student’s t -test.

    Article Snippet: Mouse monoclonal antibody (mAb) POH1 (anti-Cdc2), rabbit mAb D20 (anti-Rb), rabbit mAb against phospho-Rb (Ser807/811) D20B12, and rabbit polyclonal phospho-Cdc2 (Tyr15) antibody (product # 9111) purchased from Cell Signaling Technology (Danvers, MA).

    Techniques: Transfection, Plasmid Preparation, Staining, Flow Cytometry, Western Blot, Quantitative RT-PCR