Structured Review

Boehringer Mannheim histone h1
FKA increases MPM-2 phosphorylation and Cdc kinase activity via inhibition of Cdc2 and Cdc25 phosphorylation and downregulation of Myt1 and Wee1 expression. ( A ) Cdc2 associated <t>histone</t> H1 kinase activity was decreased dose-dependently by FKA treatment for 24 h in SKBR3 cells; ( B – D ) SKBR3 cells were treated with indicated concentrations of FKA for 24 h. Phosphorylation levels of Cdc2, MPM-2, and Cdc25C as well as protein levels of cyclin B1, Myt1, and Wee1 were detected by Western blotting analysis. β-actin was detected as a loading control. A representative blot was shown from three independent experiments.
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Images

1) Product Images from "Induction of G2M Arrest by Flavokawain A, a Kava Chalcone, Increases the Responsiveness of HER2-Overexpressing Breast Cancer Cells to Herceptin"

Article Title: Induction of G2M Arrest by Flavokawain A, a Kava Chalcone, Increases the Responsiveness of HER2-Overexpressing Breast Cancer Cells to Herceptin

Journal: Molecules (Basel, Switzerland)

doi: 10.3390/molecules22030462

FKA increases MPM-2 phosphorylation and Cdc kinase activity via inhibition of Cdc2 and Cdc25 phosphorylation and downregulation of Myt1 and Wee1 expression. ( A ) Cdc2 associated histone H1 kinase activity was decreased dose-dependently by FKA treatment for 24 h in SKBR3 cells; ( B – D ) SKBR3 cells were treated with indicated concentrations of FKA for 24 h. Phosphorylation levels of Cdc2, MPM-2, and Cdc25C as well as protein levels of cyclin B1, Myt1, and Wee1 were detected by Western blotting analysis. β-actin was detected as a loading control. A representative blot was shown from three independent experiments.
Figure Legend Snippet: FKA increases MPM-2 phosphorylation and Cdc kinase activity via inhibition of Cdc2 and Cdc25 phosphorylation and downregulation of Myt1 and Wee1 expression. ( A ) Cdc2 associated histone H1 kinase activity was decreased dose-dependently by FKA treatment for 24 h in SKBR3 cells; ( B – D ) SKBR3 cells were treated with indicated concentrations of FKA for 24 h. Phosphorylation levels of Cdc2, MPM-2, and Cdc25C as well as protein levels of cyclin B1, Myt1, and Wee1 were detected by Western blotting analysis. β-actin was detected as a loading control. A representative blot was shown from three independent experiments.

Techniques Used: Activity Assay, Inhibition, Expressing, Western Blot

2) Product Images from "Direct Activation of Cyclin-Dependent Kinase 2 by Human Papillomavirus E7"

Article Title: Direct Activation of Cyclin-Dependent Kinase 2 by Human Papillomavirus E7

Journal: Journal of Virology

doi: 10.1128/JVI.77.19.10566-10574.2003

Cross-linking of E7 peptides to CDK2/cyclin A complex. (A) HPV16 E7 (1.8 μM) and E7-derived peptides (50 μM) stimulate CDK2/cyclin A histone kinase activity. (B) Benzo-CYS-9-48-bio and benzo-NH 2 -9-48-bio peptides associate with cyclin A/CDK2 complex following UV irradiation. Histone H1 is not cross-linked under identical conditions. (C) CDK2/cyclin A (0.1 μM) and benzo-CYS-9-48-bio (0.5 μM) were with or without competitor E7 or E7 peptide. Both full-length 16E7 and the 9-48 16E7 peptide compete with the benzo-CYS-9-48-bio peptide for binding to the cyclin A/CDK2 complex. These results suggest that labeling of the CDK2/cyclin A complex by benzo-CYS-9-48-bio is specific and is not due to indiscriminate labeling of the complex. (D) Immunoprecipitation (IP) followed by Western blotting for incorporation of biotin-containing peptide into immunoprecipitated target protein(s) demonstrates that biotin-reactive bands in Western blots are cyclin A and CDK2 and that reactivity of these bands with benzo-CYS-9-48-bio peptide is dependent upon UV. (E) Cross-linking of benzophenone-containing peptides to increasing amounts of GST, cyclin A, or CDK2 demonstrates a strong preference of the peptide for cyclin A.
Figure Legend Snippet: Cross-linking of E7 peptides to CDK2/cyclin A complex. (A) HPV16 E7 (1.8 μM) and E7-derived peptides (50 μM) stimulate CDK2/cyclin A histone kinase activity. (B) Benzo-CYS-9-48-bio and benzo-NH 2 -9-48-bio peptides associate with cyclin A/CDK2 complex following UV irradiation. Histone H1 is not cross-linked under identical conditions. (C) CDK2/cyclin A (0.1 μM) and benzo-CYS-9-48-bio (0.5 μM) were with or without competitor E7 or E7 peptide. Both full-length 16E7 and the 9-48 16E7 peptide compete with the benzo-CYS-9-48-bio peptide for binding to the cyclin A/CDK2 complex. These results suggest that labeling of the CDK2/cyclin A complex by benzo-CYS-9-48-bio is specific and is not due to indiscriminate labeling of the complex. (D) Immunoprecipitation (IP) followed by Western blotting for incorporation of biotin-containing peptide into immunoprecipitated target protein(s) demonstrates that biotin-reactive bands in Western blots are cyclin A and CDK2 and that reactivity of these bands with benzo-CYS-9-48-bio peptide is dependent upon UV. (E) Cross-linking of benzophenone-containing peptides to increasing amounts of GST, cyclin A, or CDK2 demonstrates a strong preference of the peptide for cyclin A.

Techniques Used: Derivative Assay, Activity Assay, Irradiation, Binding Assay, Labeling, Immunoprecipitation, Western Blot

Addition of increasing amounts of GST-16E7 to CDK2 purified from insect cells or from bacteria results in a dose-dependent increase in kinase activity. Autoradiograph (A) demonstrates E7-dependent increase in phosphorylation of histone H1 by CDK2/cyclin A. Quantification of the experiment (B) plots PhosphorImager counts versus amount of GST-16E7 added. Results indicate that E7 affect peaks at 640 ng of E7 (or approximately 0.6 μM E7). CDK2/cyclin A reconstituted from bacterially expressed proteins is also activated by purified 16E7 (C).
Figure Legend Snippet: Addition of increasing amounts of GST-16E7 to CDK2 purified from insect cells or from bacteria results in a dose-dependent increase in kinase activity. Autoradiograph (A) demonstrates E7-dependent increase in phosphorylation of histone H1 by CDK2/cyclin A. Quantification of the experiment (B) plots PhosphorImager counts versus amount of GST-16E7 added. Results indicate that E7 affect peaks at 640 ng of E7 (or approximately 0.6 μM E7). CDK2/cyclin A reconstituted from bacterially expressed proteins is also activated by purified 16E7 (C).

Techniques Used: Purification, Activity Assay, Autoradiography

GST-16E7 activates purified cyclin E/CDK2 and cyclin A/CDK2 complexes. Autoradiographs demonstrate phosphorylation of histone H1 and Rb (pRb) by CDK2/cyclin E (A) and CDK2/cyclin A (B). (A) E7 potently upregulates CDK2/cyclin E histone H1 kinase activity while having modest effects on Rb kinase activity. Addition of p27 eliminates this effect and reduces activity to below baseline levels. (B) CDK2/cyclin A histone kinase activity is also dramatically upregulated by E7, and the effect is negated by addition of p27.
Figure Legend Snippet: GST-16E7 activates purified cyclin E/CDK2 and cyclin A/CDK2 complexes. Autoradiographs demonstrate phosphorylation of histone H1 and Rb (pRb) by CDK2/cyclin E (A) and CDK2/cyclin A (B). (A) E7 potently upregulates CDK2/cyclin E histone H1 kinase activity while having modest effects on Rb kinase activity. Addition of p27 eliminates this effect and reduces activity to below baseline levels. (B) CDK2/cyclin A histone kinase activity is also dramatically upregulated by E7, and the effect is negated by addition of p27.

Techniques Used: Purification, Activity Assay

3) Product Images from "p21Waf1/Cip1 Inhibition of Cyclin E/Cdk2 Activity Prevents Endoreduplication after Mitotic Spindle Disruption"

Article Title: p21Waf1/Cip1 Inhibition of Cyclin E/Cdk2 Activity Prevents Endoreduplication after Mitotic Spindle Disruption

Journal: Molecular and Cellular Biology

doi:

p21 induction in p53-deficient cells during MTI-induced endoreduplication reduces cyclin B1/Cdc2 kinase activity. (A) Western analysis of cyclin B1 and Cdc2. Asynchronous cells were treated with nocodazole (83 nM) for the indicated times, and protein was harvested. (B) Cyclin B1-associated kinase activity in the presence and absence of muristerone (7.5 μM) in nocodazole-treated HIp21 cells. Anti-cyclin B1 antibody was used to immunoprecipitate kinase complexes; histone H1 was used as a substrate. Quantification of the autoradiogram signals is presented in the histograms. Results are representative of three independent experiments.
Figure Legend Snippet: p21 induction in p53-deficient cells during MTI-induced endoreduplication reduces cyclin B1/Cdc2 kinase activity. (A) Western analysis of cyclin B1 and Cdc2. Asynchronous cells were treated with nocodazole (83 nM) for the indicated times, and protein was harvested. (B) Cyclin B1-associated kinase activity in the presence and absence of muristerone (7.5 μM) in nocodazole-treated HIp21 cells. Anti-cyclin B1 antibody was used to immunoprecipitate kinase complexes; histone H1 was used as a substrate. Quantification of the autoradiogram signals is presented in the histograms. Results are representative of three independent experiments.

Techniques Used: Activity Assay, Western Blot

Loss of p21 results in cyclical Cdc2 kinase activity during MTI-induced endoreduplication. (A) Western analysis of cyclin B1 and Cdc2 proteins. Asynchronous cells were treated with nocodazole (83 nM) for the indicated times, and protein was harvested. (B) Cyclin B1-associated kinase activity in HCT116 p21 +/+ and p21 −/− cells. Anti-cyclin B1 antibody was used to immunoprecipitate kinase complexes; histone H1 was used as a substrate. Quantification of the autoradiogram signals is presented in the histograms. Results are representative of three independent experiments. (C) Whole-cell lysates from HCT116 p21 +/+ cells were immunoprecipitated with anti-cyclin B1, separated by SDS-PAGE, and analyzed for coimmunoprecipitation of p21 by Western blot analysis. Con, control representing p21 protein coimmunoprecipitated with anti-cyclin B1 from a whole-cell extract of WI-38 fibroblasts.
Figure Legend Snippet: Loss of p21 results in cyclical Cdc2 kinase activity during MTI-induced endoreduplication. (A) Western analysis of cyclin B1 and Cdc2 proteins. Asynchronous cells were treated with nocodazole (83 nM) for the indicated times, and protein was harvested. (B) Cyclin B1-associated kinase activity in HCT116 p21 +/+ and p21 −/− cells. Anti-cyclin B1 antibody was used to immunoprecipitate kinase complexes; histone H1 was used as a substrate. Quantification of the autoradiogram signals is presented in the histograms. Results are representative of three independent experiments. (C) Whole-cell lysates from HCT116 p21 +/+ cells were immunoprecipitated with anti-cyclin B1, separated by SDS-PAGE, and analyzed for coimmunoprecipitation of p21 by Western blot analysis. Con, control representing p21 protein coimmunoprecipitated with anti-cyclin B1 from a whole-cell extract of WI-38 fibroblasts.

Techniques Used: Activity Assay, Western Blot, Immunoprecipitation, SDS Page

4) Product Images from "The human papillomavirus E7 oncoprotein can uncouple cellular differentiation and proliferation in human keratinocytes by abrogating p21Cip1-mediated inhibition of cdk2"

Article Title: The human papillomavirus E7 oncoprotein can uncouple cellular differentiation and proliferation in human keratinocytes by abrogating p21Cip1-mediated inhibition of cdk2

Journal: Genes & Development

doi:

The low-risk HPV-6 E7 protein can bind to p21 Cip1 and overcome p21 Cip1 -mediated inhibition of cyclin E-associated histone H1 kinase activity. ( A ) Radioactively labeled p21 Cip1 protein was synthesized by in vitro transcription/in vitro translation in the presence of [ 35 S]-methionine and mixed with purified GST-16 E7, GST-6 E7 or unfused GST as a control. Complexes were analyzed by glutathione–sepharose affinity chromatography, SDS-PAGE, and fluorography. The signal of p21 Cip1 in complex with wild-type HPV-16 E7 corresponds to ∼5% of the input protein. ( B ) Cyclin E-associated protein kinases were immunoprecipitated from the human myeloid leukemia cell line ML-1 and kinase assays were performed in the absence (−) or presence (+) of 2 μg of purified GST–p21 Cip1 using histone H1 as a substrate. Increasing amounts of purified GST–6 E7 (0.4, 1.1, 4.4, and 6.6 μg) were titrated into the reaction. As a control, purified unfused GST (1.1, 4.4, and 6.6 μg). Quantitation is shown underneath.
Figure Legend Snippet: The low-risk HPV-6 E7 protein can bind to p21 Cip1 and overcome p21 Cip1 -mediated inhibition of cyclin E-associated histone H1 kinase activity. ( A ) Radioactively labeled p21 Cip1 protein was synthesized by in vitro transcription/in vitro translation in the presence of [ 35 S]-methionine and mixed with purified GST-16 E7, GST-6 E7 or unfused GST as a control. Complexes were analyzed by glutathione–sepharose affinity chromatography, SDS-PAGE, and fluorography. The signal of p21 Cip1 in complex with wild-type HPV-16 E7 corresponds to ∼5% of the input protein. ( B ) Cyclin E-associated protein kinases were immunoprecipitated from the human myeloid leukemia cell line ML-1 and kinase assays were performed in the absence (−) or presence (+) of 2 μg of purified GST–p21 Cip1 using histone H1 as a substrate. Increasing amounts of purified GST–6 E7 (0.4, 1.1, 4.4, and 6.6 μg) were titrated into the reaction. As a control, purified unfused GST (1.1, 4.4, and 6.6 μg). Quantitation is shown underneath.

Techniques Used: Inhibition, Activity Assay, Labeling, Synthesized, In Vitro, Purification, Affinity Chromatography, SDS Page, Immunoprecipitation, Quantitation Assay

( A ) Immunoblot analysis of markers of cellular differentiation (Involucrin; Keratin 10) and proliferation (proliferating cellular nuclear antigen, PCNA; retinoblastoma tumor suppressor protein, pRB) in keratinocytes infected with a control virus (LXSN) or an HPV-16 E7-expressing retrovirus (E7) at 0, 1, 6, 24, and 72 hr after induction of differentiation. ( B ) Analysis of p21 Cip1 , expression, p21 Cip1 association with cdk2, and histone H1 (HH1) kinase activity associated with cdk2 at 0, 24, and 72 hr after induction of differentiation. To allow for quantitative comparisons, the different panels of LXSN and E7-expressing keratinocytes shown in this figure are derived from identical exposures of the same gel.
Figure Legend Snippet: ( A ) Immunoblot analysis of markers of cellular differentiation (Involucrin; Keratin 10) and proliferation (proliferating cellular nuclear antigen, PCNA; retinoblastoma tumor suppressor protein, pRB) in keratinocytes infected with a control virus (LXSN) or an HPV-16 E7-expressing retrovirus (E7) at 0, 1, 6, 24, and 72 hr after induction of differentiation. ( B ) Analysis of p21 Cip1 , expression, p21 Cip1 association with cdk2, and histone H1 (HH1) kinase activity associated with cdk2 at 0, 24, and 72 hr after induction of differentiation. To allow for quantitative comparisons, the different panels of LXSN and E7-expressing keratinocytes shown in this figure are derived from identical exposures of the same gel.

Techniques Used: Cell Differentiation, Infection, Expressing, Activity Assay, Derivative Assay

HPV-16 E7 can overcome p21 Cip1 -mediated inhibition of cyclin A- and cyclin E-associated histone H1 kinase activity in vitro. Cyclin A-associated ( A,B ) and cyclin E-associated ( C ) protein kinases were immunoprecipitated from the human myeloid leukemia cell line ML-1 and kinase assays were performed in the absence (−) or presence (+) of 2 μg of purified GST–p21 Cip1 using histone H1 as a substrate. Increasing amounts of purified GST–16E7 (0.4, 1.1, 4.4, and 6.6 μg) ( A,C ) or the pRB-binding deficient mutant GST–16E7Δ21-24 (0.4, 1.1, 4.4, and 6.6 μg) ( B ) were titrated into the reaction. As a control, purified unfused GST (1.1, 4.4, and 6.6 μg) was added. Quantitations are shown underneath each assay.
Figure Legend Snippet: HPV-16 E7 can overcome p21 Cip1 -mediated inhibition of cyclin A- and cyclin E-associated histone H1 kinase activity in vitro. Cyclin A-associated ( A,B ) and cyclin E-associated ( C ) protein kinases were immunoprecipitated from the human myeloid leukemia cell line ML-1 and kinase assays were performed in the absence (−) or presence (+) of 2 μg of purified GST–p21 Cip1 using histone H1 as a substrate. Increasing amounts of purified GST–16E7 (0.4, 1.1, 4.4, and 6.6 μg) ( A,C ) or the pRB-binding deficient mutant GST–16E7Δ21-24 (0.4, 1.1, 4.4, and 6.6 μg) ( B ) were titrated into the reaction. As a control, purified unfused GST (1.1, 4.4, and 6.6 μg) was added. Quantitations are shown underneath each assay.

Techniques Used: Inhibition, Activity Assay, In Vitro, Immunoprecipitation, Purification, Binding Assay, Mutagenesis

5) Product Images from "Identification of a substrate-targeting domain in cyclin E necessary for phosphorylation of the retinoblastoma protein"

Article Title: Identification of a substrate-targeting domain in cyclin E necessary for phosphorylation of the retinoblastoma protein

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi:

Transfection of wild-type and mutant cyclin E proteins into SAOS-2 cells. ( A ) Percentage of cells in G 1 phase of the cell cycle versus amount of transfected plasmid. ( B ) SAOS-2 cells transfected with pCMVpRb plasmid and increasing amounts of cyclin E: percentage of cells in G 1 phase of the cell cycle versus amount of transfected plasmid. ( C ) Cyclin E-associated histone H1 kinase activity from cells transfected with pCMVpRb plus cyclin E ( D ) Phosphorylation of transfected full-length Rb by cyclin E (5 μg of transfected DNA) in SAOS-2 cells.
Figure Legend Snippet: Transfection of wild-type and mutant cyclin E proteins into SAOS-2 cells. ( A ) Percentage of cells in G 1 phase of the cell cycle versus amount of transfected plasmid. ( B ) SAOS-2 cells transfected with pCMVpRb plasmid and increasing amounts of cyclin E: percentage of cells in G 1 phase of the cell cycle versus amount of transfected plasmid. ( C ) Cyclin E-associated histone H1 kinase activity from cells transfected with pCMVpRb plus cyclin E ( D ) Phosphorylation of transfected full-length Rb by cyclin E (5 μg of transfected DNA) in SAOS-2 cells.

Techniques Used: Transfection, Mutagenesis, Plasmid Preparation, Activity Assay

VxCxE mutants are defective for Rb but not histone H1 phosphorylation in vitro . ( A ) Recombinant cyclin E–CDK2 complexes made in baculovirus-infected Sf-9 insect cells were incubated with [γ- 32 P]ATP and either histone H1 ( A ) or Rb ( B ) for increasing amounts of time. Results shown are representative of three different experiments.
Figure Legend Snippet: VxCxE mutants are defective for Rb but not histone H1 phosphorylation in vitro . ( A ) Recombinant cyclin E–CDK2 complexes made in baculovirus-infected Sf-9 insect cells were incubated with [γ- 32 P]ATP and either histone H1 ( A ) or Rb ( B ) for increasing amounts of time. Results shown are representative of three different experiments.

Techniques Used: In Vitro, Recombinant, Infection, Incubation

Transfection of wild-type and mutant cyclin E proteins into NIH 3T3 cells. NIH 3T3 cells were transiently transfected with increasing amounts of myc-tagged wild-type or mutant cyclin E. ( A ) Percentage of cells in G 1 phase of the cell cycle versus amount of transfected plasmid. ( B ) Cyclin E protein expression and associated histone H1 kinase activity from cells depicted in A.
Figure Legend Snippet: Transfection of wild-type and mutant cyclin E proteins into NIH 3T3 cells. NIH 3T3 cells were transiently transfected with increasing amounts of myc-tagged wild-type or mutant cyclin E. ( A ) Percentage of cells in G 1 phase of the cell cycle versus amount of transfected plasmid. ( B ) Cyclin E protein expression and associated histone H1 kinase activity from cells depicted in A.

Techniques Used: Transfection, Mutagenesis, Plasmid Preparation, Expressing, Activity Assay

6) Product Images from "Role of the casein kinase I isoform, Hrr25, and the cell cycle-regulatory transcription factor, SBF, in the transcriptional response to DNA damage in Saccharomyces cerevisiae"

Article Title: Role of the casein kinase I isoform, Hrr25, and the cell cycle-regulatory transcription factor, SBF, in the transcriptional response to DNA damage in Saccharomyces cerevisiae

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi:

In vitro  phosphorylation of Swi6 by Hrr25. ( A ) Kinase assays were done on eluates from Swi6 affinity columns or from control columns (no coupled protein). The presence of Swi6 on the column resin is indicated by a “+” above the lane, whereas “−” denotes the control column with no coupled protein. The columns were loaded with extracts from either a wild-type (lanes 1–7) or  hrr25 Δ strain (lanes 8 and 9) as indicated above the figure (“extract applied on column”). Exogenous substrate (100 ng) was added to the kinase assays as indicated above the lanes. mbp, myelin basic protein; H1, histone H1. ( B ) Kinase assays were done with 12CA5 (anti-HA) immunoprecipitates from yeast cells expressing an HA–Hrr25 fusion protein (lanes 1 and 3) or cells transformed with an empty vector (lane 2). In lanes 2 and 3, 100 ng of casein and Swi6 were added to the kinase reaction as indicated by a “+.” The position of migration of phosphorylated Swi6, Hrr25, and casein are indicated on the right. Molecular weight markers are shown on the left.
Figure Legend Snippet: In vitro phosphorylation of Swi6 by Hrr25. ( A ) Kinase assays were done on eluates from Swi6 affinity columns or from control columns (no coupled protein). The presence of Swi6 on the column resin is indicated by a “+” above the lane, whereas “−” denotes the control column with no coupled protein. The columns were loaded with extracts from either a wild-type (lanes 1–7) or hrr25 Δ strain (lanes 8 and 9) as indicated above the figure (“extract applied on column”). Exogenous substrate (100 ng) was added to the kinase assays as indicated above the lanes. mbp, myelin basic protein; H1, histone H1. ( B ) Kinase assays were done with 12CA5 (anti-HA) immunoprecipitates from yeast cells expressing an HA–Hrr25 fusion protein (lanes 1 and 3) or cells transformed with an empty vector (lane 2). In lanes 2 and 3, 100 ng of casein and Swi6 were added to the kinase reaction as indicated by a “+.” The position of migration of phosphorylated Swi6, Hrr25, and casein are indicated on the right. Molecular weight markers are shown on the left.

Techniques Used: In Vitro, Expressing, Transformation Assay, Plasmid Preparation, Migration, Molecular Weight

7) Product Images from "Posttranslational Processing of Infected Cell Proteins 0 and 4 of Herpes Simplex Virus 1 Is Sequential and Reflects the Subcellular Compartment in Which the Proteins Localize"

Article Title: Posttranslational Processing of Infected Cell Proteins 0 and 4 of Herpes Simplex Virus 1 Is Sequential and Reflects the Subcellular Compartment in Which the Proteins Localize

Journal: Journal of Virology

doi: 10.1128/JVI.75.17.7904-7912.2001

Autoradiograms of histone H1 phosphorylated by in vitro kinase assays using immunoprecipitated cdk2 or cdc2 from HSV-1- or mock-infected cells. HEp-2 cells were HSV-1 or mock infected; 5 h after infection, the medium was replaced with medium containing 0, 10, or 100 μM roscovitine. Cells were harvested 12 h after infection, and cdk2 or cdc2 was immunoprecipitated (IP) with antibodie (Ab) as described in Materials and Methods. Kinase assays were done using histone H1 as the substrate. Phosphorylated histone H1 was quantitated by a PhosphorImager. Relative reduction in the phosphorylation of histone H1 due to roscovitine was determined relative to untreated samples.
Figure Legend Snippet: Autoradiograms of histone H1 phosphorylated by in vitro kinase assays using immunoprecipitated cdk2 or cdc2 from HSV-1- or mock-infected cells. HEp-2 cells were HSV-1 or mock infected; 5 h after infection, the medium was replaced with medium containing 0, 10, or 100 μM roscovitine. Cells were harvested 12 h after infection, and cdk2 or cdc2 was immunoprecipitated (IP) with antibodie (Ab) as described in Materials and Methods. Kinase assays were done using histone H1 as the substrate. Phosphorylated histone H1 was quantitated by a PhosphorImager. Relative reduction in the phosphorylation of histone H1 due to roscovitine was determined relative to untreated samples.

Techniques Used: In Vitro, Immunoprecipitation, Infection

8) Product Images from "Transgene Expression of bcl-xL Permits Anti-immunoglobulin (Ig)-induced Proliferation in xid B Cells "

Article Title: Transgene Expression of bcl-xL Permits Anti-immunoglobulin (Ig)-induced Proliferation in xid B Cells

Journal: The Journal of Experimental Medicine

doi:

In vitro kinase assays from anti-IgM–stimulated B cells. Stimulated B cells were collected at the time points indicated and used to measure ( A ) cdk2- and ( B ) cdc2-associated specific kinase activity using Histone H1 as the substrate in the presence of [ 32 P]gATP. Kinase activity was quantitated by comparing the amount of 32 P incorporated into the substrate by phosphorimager analysis. Kinase activity detected in xid ( black bars ) and CBA/Ca ( gray bars ) are shown as arbitrary units. Similar results were obtained in three independent experiments.
Figure Legend Snippet: In vitro kinase assays from anti-IgM–stimulated B cells. Stimulated B cells were collected at the time points indicated and used to measure ( A ) cdk2- and ( B ) cdc2-associated specific kinase activity using Histone H1 as the substrate in the presence of [ 32 P]gATP. Kinase activity was quantitated by comparing the amount of 32 P incorporated into the substrate by phosphorimager analysis. Kinase activity detected in xid ( black bars ) and CBA/Ca ( gray bars ) are shown as arbitrary units. Similar results were obtained in three independent experiments.

Techniques Used: In Vitro, Activity Assay, Crocin Bleaching Assay

9) Product Images from "Intracellular colocalization and interaction of IGF-binding protein-2 with the cyclin-dependent kinase inhibitor p21CIP1/WAF1 during growth inhibition"

Article Title: Intracellular colocalization and interaction of IGF-binding protein-2 with the cyclin-dependent kinase inhibitor p21CIP1/WAF1 during growth inhibition

Journal: Biochemical Journal

doi: 10.1042/BJ20050517

p21 CIP1/WAF1 expression and CDK activity in growth-inhibited MLE-12 cells Sub-confluent MLE-12 cells were cultured in HITES medium, in the presence of serum for 24 h (CTL) or in the absence of serum (0% FBS) for 24, 48 or 72 h. ( A ) Western blot (WB) analysis of cytoplasmic and nuclear p21 CIP1/WAF1 protein. Equal amounts of cytoplasmic and nuclear protein extracts from control cells (CTL) and from serum-deprived cells were analysed by Western blotting with anti-p21 CIP1/WAF1 antibody. Antibodies against β-actin and TATA-binding protein (α-TBP) were used as controls for protein loading in the cytoplasmic and nuclear extracts respectively. The histogram shows a quantitative representation of cytoplasmic and nuclear levels of p21 CIP1/WAF1 protein obtained from laser densitometric analysis of three independent experiments. ( B ) CDK activity of cell cycle proteins. Cyclin E and cyclin A were immunoprecipitated and associated kinase activity was assayed using histone H1 as substrate. Equal amounts of nuclear extracts from control cells (CTL) or from serum-deprived cells were used. The histogram shows a quantitative representation obtained from laser densitometric analysis of three independent experiments. ( C ) Western blot analysis of cell cycle proteins. Cyclin E and cyclin A were detected by Western blot analysis with appropriate antisera. Equal amounts of nuclear extracts from control cells (CTL) or from serum-deprived cells were used. The histogram shows a quantitative representation obtained from laser densitometric analysis of three independent experiments. All densitometry results were expressed as fold induction. * and #, P
Figure Legend Snippet: p21 CIP1/WAF1 expression and CDK activity in growth-inhibited MLE-12 cells Sub-confluent MLE-12 cells were cultured in HITES medium, in the presence of serum for 24 h (CTL) or in the absence of serum (0% FBS) for 24, 48 or 72 h. ( A ) Western blot (WB) analysis of cytoplasmic and nuclear p21 CIP1/WAF1 protein. Equal amounts of cytoplasmic and nuclear protein extracts from control cells (CTL) and from serum-deprived cells were analysed by Western blotting with anti-p21 CIP1/WAF1 antibody. Antibodies against β-actin and TATA-binding protein (α-TBP) were used as controls for protein loading in the cytoplasmic and nuclear extracts respectively. The histogram shows a quantitative representation of cytoplasmic and nuclear levels of p21 CIP1/WAF1 protein obtained from laser densitometric analysis of three independent experiments. ( B ) CDK activity of cell cycle proteins. Cyclin E and cyclin A were immunoprecipitated and associated kinase activity was assayed using histone H1 as substrate. Equal amounts of nuclear extracts from control cells (CTL) or from serum-deprived cells were used. The histogram shows a quantitative representation obtained from laser densitometric analysis of three independent experiments. ( C ) Western blot analysis of cell cycle proteins. Cyclin E and cyclin A were detected by Western blot analysis with appropriate antisera. Equal amounts of nuclear extracts from control cells (CTL) or from serum-deprived cells were used. The histogram shows a quantitative representation obtained from laser densitometric analysis of three independent experiments. All densitometry results were expressed as fold induction. * and #, P

Techniques Used: Expressing, Activity Assay, Cell Culture, CTL Assay, Western Blot, Binding Assay, Immunoprecipitation

10) Product Images from "Phosphorylation- and ubiquitin-dependent degradation of the cyclin-dependent kinase inhibitor Far1p in budding yeast"

Article Title: Phosphorylation- and ubiquitin-dependent degradation of the cyclin-dependent kinase inhibitor Far1p in budding yeast

Journal: Genes & Development

doi:

Far1p is phosphorylated by Cdc28p–Cln2p kinase on Ser-87 in vitro. ( A ) Full-length wild-type Far1p was produced in E. coli, purified as a GST fusion protein and phosphorylated in vitro with Cdc28p–Cln2p kinase immunoprecipitated from yeast extracts (lane 3 ). As a control, the kinase assays were performed with GST alone (lane 2 ) or without substrate (lane 1 ). Phosphorylated proteins were analyzed by autoradiography ( top left panel) and immunoblotting with specific antibodies against Far1p ( bottom left panel). The arrowhead marks the position of GST-Far1 protein. A fragment containing amino acids 50–340 of either wild-type Far1p (lane 4 ) or Far1-22p (lane 5 ) was expressed as a GST fusion protein in E. coli and phosphorylated in vitro by immunoprecipitated Cln2–HAp-associated kinase. Kinase reactions were also carried out with histone H1 (lane 7 ) or in the absence of substrate (lane 6 ). The specificity of the immunoprecipitated Cln2p-associated kinase was confirmed by including an untagged Cln2p (lane 8 ). The addition of equal amounts of GST–Far1p in the kinase reactions was verified by immunoblotting with antibodies against Far1p ( bottom right panel). The arrowhead points to the position of the GST-Far1 50-340 protein; the bracket marks the position of phosphorylated histone H1. ( B ) Sequencing of the FAR1-22 ). ( C ) Ser-87 is phosphorylated by Cdc28p–Cln2p kinase in vitro. A fragment containing amino acids 50–340 of either wild-type Far1p ( top panel) or Far1p-S87T, in which Ser-87 was replaced by a threonine residue ( bottom panel), was phosphorylated in vitro by immunoprecipitated Cln2–HAp-associated kinase and phosphoamino acid analysis was performed. (S) The position of phosphoserine; (T) The position of phosphothreonine.
Figure Legend Snippet: Far1p is phosphorylated by Cdc28p–Cln2p kinase on Ser-87 in vitro. ( A ) Full-length wild-type Far1p was produced in E. coli, purified as a GST fusion protein and phosphorylated in vitro with Cdc28p–Cln2p kinase immunoprecipitated from yeast extracts (lane 3 ). As a control, the kinase assays were performed with GST alone (lane 2 ) or without substrate (lane 1 ). Phosphorylated proteins were analyzed by autoradiography ( top left panel) and immunoblotting with specific antibodies against Far1p ( bottom left panel). The arrowhead marks the position of GST-Far1 protein. A fragment containing amino acids 50–340 of either wild-type Far1p (lane 4 ) or Far1-22p (lane 5 ) was expressed as a GST fusion protein in E. coli and phosphorylated in vitro by immunoprecipitated Cln2–HAp-associated kinase. Kinase reactions were also carried out with histone H1 (lane 7 ) or in the absence of substrate (lane 6 ). The specificity of the immunoprecipitated Cln2p-associated kinase was confirmed by including an untagged Cln2p (lane 8 ). The addition of equal amounts of GST–Far1p in the kinase reactions was verified by immunoblotting with antibodies against Far1p ( bottom right panel). The arrowhead points to the position of the GST-Far1 50-340 protein; the bracket marks the position of phosphorylated histone H1. ( B ) Sequencing of the FAR1-22 ). ( C ) Ser-87 is phosphorylated by Cdc28p–Cln2p kinase in vitro. A fragment containing amino acids 50–340 of either wild-type Far1p ( top panel) or Far1p-S87T, in which Ser-87 was replaced by a threonine residue ( bottom panel), was phosphorylated in vitro by immunoprecipitated Cln2–HAp-associated kinase and phosphoamino acid analysis was performed. (S) The position of phosphoserine; (T) The position of phosphothreonine.

Techniques Used: In Vitro, Produced, Purification, Immunoprecipitation, Autoradiography, Sequencing, Phosphoamino Acid Analysis

Cells producing Far1-22p arrest in G 1 by inhibition of the G 1 kinase, Cdc28p–Clnp. ( A,B ) Cln2p tagged at its carboxyl terminus with three copies of the HA epitope (Cln2–HA) was immunoprecipitated from extracts prepared from cells (YMT263) expressing either Far1-22p or for control, an inactive mutant form of Far1p, Far1-22/Δ285–350, from the inducible GAL promoter. Cells were grown in media containing galactose (GAL, GAL promoter on) or glucose (GLU, GAL promoter off). Cln2–HAp-associated kinase activity was measured with histone H1 as a substrate and quantified ( A ). The kinase activity associated with Cln2–HAp from cells grown in glucose was normalized to 100%. Similar amounts of Cln2–HAp were immunoprecipitated in each assay as shown by immunoblotting ( B ). ( C ), and Far1-22/Δ285–350. ( D ) Growth inhibition caused by expression of Far1-22p was rescued by co-overexpression of a stable G 1 -cyclin, Cln3p ( DAF1 ). DAF1-1 cells (IH2517), which express a stable form of the G 1 cyclin Cln3p or isogenic wild-type cells (IH2518), were transformed with plasmids expressing either wild-type Far1p, Far1-22p, or Far1-22/Δ285–350p from the inducible GAL promoter.
Figure Legend Snippet: Cells producing Far1-22p arrest in G 1 by inhibition of the G 1 kinase, Cdc28p–Clnp. ( A,B ) Cln2p tagged at its carboxyl terminus with three copies of the HA epitope (Cln2–HA) was immunoprecipitated from extracts prepared from cells (YMT263) expressing either Far1-22p or for control, an inactive mutant form of Far1p, Far1-22/Δ285–350, from the inducible GAL promoter. Cells were grown in media containing galactose (GAL, GAL promoter on) or glucose (GLU, GAL promoter off). Cln2–HAp-associated kinase activity was measured with histone H1 as a substrate and quantified ( A ). The kinase activity associated with Cln2–HAp from cells grown in glucose was normalized to 100%. Similar amounts of Cln2–HAp were immunoprecipitated in each assay as shown by immunoblotting ( B ). ( C ), and Far1-22/Δ285–350. ( D ) Growth inhibition caused by expression of Far1-22p was rescued by co-overexpression of a stable G 1 -cyclin, Cln3p ( DAF1 ). DAF1-1 cells (IH2517), which express a stable form of the G 1 cyclin Cln3p or isogenic wild-type cells (IH2518), were transformed with plasmids expressing either wild-type Far1p, Far1-22p, or Far1-22/Δ285–350p from the inducible GAL promoter.

Techniques Used: Inhibition, Immunoprecipitation, Expressing, Mutagenesis, Activity Assay, Over Expression, Transformation Assay

11) Product Images from "c-Myc Regulates Cyclin D-Cdk4 and -Cdk6 Activity but Affects Cell Cycle Progression at Multiple Independent Points"

Article Title: c-Myc Regulates Cyclin D-Cdk4 and -Cdk6 Activity but Affects Cell Cycle Progression at Multiple Independent Points

Journal: Molecular and Cellular Biology

doi:

Cdk activities and expression of cell cycle effectors in exponentially growing cells. (A) Cdk activities. Complexes were immunoprecipitated from extracts with the antibodies indicated at the bottom. The substrate used in the kinase assays (histone H1 or Rb) is indicated in parentheses. Cell lines: TGR-1, c- myc +/+ ; HO15.19, c- myc −/− ; HOmyc3, HO15.19 with reconstituted c-Myc expression. All data are normalized to the activity measured in TGR-1 immunoprecipitates. CycA, cyclin A. Error bars indicate standard deviations of a minimum of two independent experiments. (B) Immunoblots of cyclin, Cdk, CKI, and Mad proteins. myc rec., HOmyc3.
Figure Legend Snippet: Cdk activities and expression of cell cycle effectors in exponentially growing cells. (A) Cdk activities. Complexes were immunoprecipitated from extracts with the antibodies indicated at the bottom. The substrate used in the kinase assays (histone H1 or Rb) is indicated in parentheses. Cell lines: TGR-1, c- myc +/+ ; HO15.19, c- myc −/− ; HOmyc3, HO15.19 with reconstituted c-Myc expression. All data are normalized to the activity measured in TGR-1 immunoprecipitates. CycA, cyclin A. Error bars indicate standard deviations of a minimum of two independent experiments. (B) Immunoblots of cyclin, Cdk, CKI, and Mad proteins. myc rec., HOmyc3.

Techniques Used: Expressing, Immunoprecipitation, Activity Assay, Western Blot

Cdk activities during the G 0 -to-S transition. (A) Cyclin D1 immunoprecipitates (IP). (B) Cyclin D3 immunoprecipitates. (C) Cyclin E immunoprecipitates. (D) Cdk2 immunoprecipitates. Complexes were immunoprecipitated from extracts containing equal amounts of total protein, and kinase assays were performed with either a GST-Rb substrate (A and B) or a histone H1 substrate (C and D) as described in Materials and Methods. Cell lines: TGR-1, c- myc +/+ ; HO15.19, c- myc −/− . The fold induction is expressed relative to the TGR-1 zero time point. The kinase assays shown are representative of three independent experiments.
Figure Legend Snippet: Cdk activities during the G 0 -to-S transition. (A) Cyclin D1 immunoprecipitates (IP). (B) Cyclin D3 immunoprecipitates. (C) Cyclin E immunoprecipitates. (D) Cdk2 immunoprecipitates. Complexes were immunoprecipitated from extracts containing equal amounts of total protein, and kinase assays were performed with either a GST-Rb substrate (A and B) or a histone H1 substrate (C and D) as described in Materials and Methods. Cell lines: TGR-1, c- myc +/+ ; HO15.19, c- myc −/− . The fold induction is expressed relative to the TGR-1 zero time point. The kinase assays shown are representative of three independent experiments.

Techniques Used: Immunoprecipitation

Activation of Cdk2 complexes with CAK. Cdk2 complexes were immunoprecipitated from exponentially growing cells with Cdk2 antibodies, incubated with active Cak1p, and assayed for histone H1 phosphorylation activity, as described in Materials and Methods. Cell lines: TGR-1, c- myc +/+ ; HO15.19, c- myc −/− ; HOmyc3, HO15.19 with reconstituted c-Myc expression (myc reconstr.). All data are represented as percentages of the activity measured in TGR-1 immunoprecipitates without CAK addition.
Figure Legend Snippet: Activation of Cdk2 complexes with CAK. Cdk2 complexes were immunoprecipitated from exponentially growing cells with Cdk2 antibodies, incubated with active Cak1p, and assayed for histone H1 phosphorylation activity, as described in Materials and Methods. Cell lines: TGR-1, c- myc +/+ ; HO15.19, c- myc −/− ; HOmyc3, HO15.19 with reconstituted c-Myc expression (myc reconstr.). All data are represented as percentages of the activity measured in TGR-1 immunoprecipitates without CAK addition.

Techniques Used: Activation Assay, Immunoprecipitation, Incubation, Activity Assay, Expressing

12) Product Images from "c-Myc Regulates Cyclin D-Cdk4 and -Cdk6 Activity but Affects Cell Cycle Progression at Multiple Independent Points"

Article Title: c-Myc Regulates Cyclin D-Cdk4 and -Cdk6 Activity but Affects Cell Cycle Progression at Multiple Independent Points

Journal: Molecular and Cellular Biology

doi:

Cdk activities and expression of cell cycle effectors in exponentially growing cells. (A) Cdk activities. Complexes were immunoprecipitated from extracts with the antibodies indicated at the bottom. The substrate used in the kinase assays (histone H1 or Rb) is indicated in parentheses. Cell lines: TGR-1, c- myc +/+ ; HO15.19, c- myc −/− ; HOmyc3, HO15.19 with reconstituted c-Myc expression. All data are normalized to the activity measured in TGR-1 immunoprecipitates. CycA, cyclin A. Error bars indicate standard deviations of a minimum of two independent experiments. (B) Immunoblots of cyclin, Cdk, CKI, and Mad proteins. myc rec., HOmyc3.
Figure Legend Snippet: Cdk activities and expression of cell cycle effectors in exponentially growing cells. (A) Cdk activities. Complexes were immunoprecipitated from extracts with the antibodies indicated at the bottom. The substrate used in the kinase assays (histone H1 or Rb) is indicated in parentheses. Cell lines: TGR-1, c- myc +/+ ; HO15.19, c- myc −/− ; HOmyc3, HO15.19 with reconstituted c-Myc expression. All data are normalized to the activity measured in TGR-1 immunoprecipitates. CycA, cyclin A. Error bars indicate standard deviations of a minimum of two independent experiments. (B) Immunoblots of cyclin, Cdk, CKI, and Mad proteins. myc rec., HOmyc3.

Techniques Used: Expressing, Immunoprecipitation, Activity Assay, Western Blot

Cdk activities during the G 0 -to-S transition. (A) Cyclin D1 immunoprecipitates (IP). (B) Cyclin D3 immunoprecipitates. (C) Cyclin E immunoprecipitates. (D) Cdk2 immunoprecipitates. Complexes were immunoprecipitated from extracts containing equal amounts of total protein, and kinase assays were performed with either a GST-Rb substrate (A and B) or a histone H1 substrate (C and D) as described in Materials and Methods. Cell lines: TGR-1, c- myc +/+ ; HO15.19, c- myc −/− . The fold induction is expressed relative to the TGR-1 zero time point. The kinase assays shown are representative of three independent experiments.
Figure Legend Snippet: Cdk activities during the G 0 -to-S transition. (A) Cyclin D1 immunoprecipitates (IP). (B) Cyclin D3 immunoprecipitates. (C) Cyclin E immunoprecipitates. (D) Cdk2 immunoprecipitates. Complexes were immunoprecipitated from extracts containing equal amounts of total protein, and kinase assays were performed with either a GST-Rb substrate (A and B) or a histone H1 substrate (C and D) as described in Materials and Methods. Cell lines: TGR-1, c- myc +/+ ; HO15.19, c- myc −/− . The fold induction is expressed relative to the TGR-1 zero time point. The kinase assays shown are representative of three independent experiments.

Techniques Used: Immunoprecipitation

Activation of Cdk2 complexes with CAK. Cdk2 complexes were immunoprecipitated from exponentially growing cells with Cdk2 antibodies, incubated with active Cak1p, and assayed for histone H1 phosphorylation activity, as described in Materials and Methods. Cell lines: TGR-1, c- myc +/+ ; HO15.19, c- myc −/− ; HOmyc3, HO15.19 with reconstituted c-Myc expression (myc reconstr.). All data are represented as percentages of the activity measured in TGR-1 immunoprecipitates without CAK addition.
Figure Legend Snippet: Activation of Cdk2 complexes with CAK. Cdk2 complexes were immunoprecipitated from exponentially growing cells with Cdk2 antibodies, incubated with active Cak1p, and assayed for histone H1 phosphorylation activity, as described in Materials and Methods. Cell lines: TGR-1, c- myc +/+ ; HO15.19, c- myc −/− ; HOmyc3, HO15.19 with reconstituted c-Myc expression (myc reconstr.). All data are represented as percentages of the activity measured in TGR-1 immunoprecipitates without CAK addition.

Techniques Used: Activation Assay, Immunoprecipitation, Incubation, Activity Assay, Expressing

13) Product Images from "Varicella-Zoster Virus Fc Receptor Component gI Is Phosphorylated on Its Endodomain by a Cyclin-Dependent Kinase"

Article Title: Varicella-Zoster Virus Fc Receptor Component gI Is Phosphorylated on Its Endodomain by a Cyclin-Dependent Kinase

Journal: Journal of Virology

doi:

Phosphorylation of the fusion protein GST-gI by CDK1 and CDK2. The coupled immunoprecipitation kinase assay was performed as described in Materials and Methods. The constituents in each assay are listed in tabular format above each lane at the top of the figure (+, present; −, absent). CDK2 was present in lanes 2 to 4; CDK1 was present in lanes 6 to 8. Histone H1 was the artificial substrate for the positive-control assays in lanes 4 and 8. The negative-control lanes 1 and 5 contain only GST-gI protein. For the competition assay, antibodies against CDK2 or CDK1 were preincubated for 10 min with an epitope peptide of CDK2 (lane 3) or CDK1 (lane 7). The locations of GST-gI and histone are indicated in the right margin. Molecular mass markers are on the left.
Figure Legend Snippet: Phosphorylation of the fusion protein GST-gI by CDK1 and CDK2. The coupled immunoprecipitation kinase assay was performed as described in Materials and Methods. The constituents in each assay are listed in tabular format above each lane at the top of the figure (+, present; −, absent). CDK2 was present in lanes 2 to 4; CDK1 was present in lanes 6 to 8. Histone H1 was the artificial substrate for the positive-control assays in lanes 4 and 8. The negative-control lanes 1 and 5 contain only GST-gI protein. For the competition assay, antibodies against CDK2 or CDK1 were preincubated for 10 min with an epitope peptide of CDK2 (lane 3) or CDK1 (lane 7). The locations of GST-gI and histone are indicated in the right margin. Molecular mass markers are on the left.

Techniques Used: Immunoprecipitation, Kinase Assay, Positive Control, Negative Control, Competitive Binding Assay

14) Product Images from "Fission Yeast Bub1 Is a Mitotic Centromere Protein Essential for the Spindle Checkpoint and the Preservation of Correct Ploidy through Mitosis "

Article Title: Fission Yeast Bub1 Is a Mitotic Centromere Protein Essential for the Spindle Checkpoint and the Preservation of Correct Ploidy through Mitosis

Journal: The Journal of Cell Biology

doi:

bub1 + is required for spindle checkpoint function. ( A ) bub1 mutants are hypersensitive to benomyl. Strains were grown in YES medium to 0.5–2 × 10 7 cells/ml. About 200 cells were spotted onto YES plates containing the indicated concentrations of the anti-microtubule drug benomyl and incubated at 32°C for 3 d. ( B–D ) bub1 mutants are unable to maintain a mitotic arrest. Small G2 cells of nda3KM311 Δswi6 and nda3KM311 Δbub1 were shifted to 18°C, which disrupts spindle microtubules due to the nda3 defect, and samples were taken every hour. ( B ) Unlike the Δswi6 cells which arrest with high levels of histone H1 kinase activity ( left ), the Δbub1 cells fail to maintain such an arrest and the kinase activity drops after 3 h ( right ). DAPI and calcofluor staining shows that this is followed by a wave of septation ( C ) from 4–6 h as the Δbub1 cells ( solid squares ) progress through the cell cycle, and ultimately leads to a cut phenotype ( D ) as septation occurs without chromosome segregation. The Δswi6 cells maintain their arrest with hypercondensed chromosomes (data not shown) and do not septate ( C , open circles ). ( E ) Δbub1 cells are unable to prevent sister chromatid separation in the absence of a spindle. Cells from strain 379 ( nda3KM311 ) and 401 ( nda3KM311 Δbub1 ) were grown in YES medium at 32°C to 3 × 10 6 cells/ml, shifted to 18°C for 8 h, and then fixed and processed for FISH using a probe detecting all three centromeres (cenFISH). Nuclear DNA was stained with DAPI. Bar, 5 μm.
Figure Legend Snippet: bub1 + is required for spindle checkpoint function. ( A ) bub1 mutants are hypersensitive to benomyl. Strains were grown in YES medium to 0.5–2 × 10 7 cells/ml. About 200 cells were spotted onto YES plates containing the indicated concentrations of the anti-microtubule drug benomyl and incubated at 32°C for 3 d. ( B–D ) bub1 mutants are unable to maintain a mitotic arrest. Small G2 cells of nda3KM311 Δswi6 and nda3KM311 Δbub1 were shifted to 18°C, which disrupts spindle microtubules due to the nda3 defect, and samples were taken every hour. ( B ) Unlike the Δswi6 cells which arrest with high levels of histone H1 kinase activity ( left ), the Δbub1 cells fail to maintain such an arrest and the kinase activity drops after 3 h ( right ). DAPI and calcofluor staining shows that this is followed by a wave of septation ( C ) from 4–6 h as the Δbub1 cells ( solid squares ) progress through the cell cycle, and ultimately leads to a cut phenotype ( D ) as septation occurs without chromosome segregation. The Δswi6 cells maintain their arrest with hypercondensed chromosomes (data not shown) and do not septate ( C , open circles ). ( E ) Δbub1 cells are unable to prevent sister chromatid separation in the absence of a spindle. Cells from strain 379 ( nda3KM311 ) and 401 ( nda3KM311 Δbub1 ) were grown in YES medium at 32°C to 3 × 10 6 cells/ml, shifted to 18°C for 8 h, and then fixed and processed for FISH using a probe detecting all three centromeres (cenFISH). Nuclear DNA was stained with DAPI. Bar, 5 μm.

Techniques Used: Incubation, Activity Assay, Staining, Fluorescence In Situ Hybridization

15) Product Images from "Direct Activation of Cyclin-Dependent Kinase 2 by Human Papillomavirus E7"

Article Title: Direct Activation of Cyclin-Dependent Kinase 2 by Human Papillomavirus E7

Journal: Journal of Virology

doi: 10.1128/JVI.77.19.10566-10574.2003

Cross-linking of E7 peptides to CDK2/cyclin A complex. (A) HPV16 E7 (1.8 μM) and E7-derived peptides (50 μM) stimulate CDK2/cyclin A histone kinase activity. (B) Benzo-CYS-9-48-bio and benzo-NH 2 -9-48-bio peptides associate with cyclin A/CDK2 complex following UV irradiation. Histone H1 is not cross-linked under identical conditions. (C) CDK2/cyclin A (0.1 μM) and benzo-CYS-9-48-bio (0.5 μM) were with or without competitor E7 or E7 peptide. Both full-length 16E7 and the 9-48 16E7 peptide compete with the benzo-CYS-9-48-bio peptide for binding to the cyclin A/CDK2 complex. These results suggest that labeling of the CDK2/cyclin A complex by benzo-CYS-9-48-bio is specific and is not due to indiscriminate labeling of the complex. (D) Immunoprecipitation (IP) followed by Western blotting for incorporation of biotin-containing peptide into immunoprecipitated target protein(s) demonstrates that biotin-reactive bands in Western blots are cyclin A and CDK2 and that reactivity of these bands with benzo-CYS-9-48-bio peptide is dependent upon UV. (E) Cross-linking of benzophenone-containing peptides to increasing amounts of GST, cyclin A, or CDK2 demonstrates a strong preference of the peptide for cyclin A.
Figure Legend Snippet: Cross-linking of E7 peptides to CDK2/cyclin A complex. (A) HPV16 E7 (1.8 μM) and E7-derived peptides (50 μM) stimulate CDK2/cyclin A histone kinase activity. (B) Benzo-CYS-9-48-bio and benzo-NH 2 -9-48-bio peptides associate with cyclin A/CDK2 complex following UV irradiation. Histone H1 is not cross-linked under identical conditions. (C) CDK2/cyclin A (0.1 μM) and benzo-CYS-9-48-bio (0.5 μM) were with or without competitor E7 or E7 peptide. Both full-length 16E7 and the 9-48 16E7 peptide compete with the benzo-CYS-9-48-bio peptide for binding to the cyclin A/CDK2 complex. These results suggest that labeling of the CDK2/cyclin A complex by benzo-CYS-9-48-bio is specific and is not due to indiscriminate labeling of the complex. (D) Immunoprecipitation (IP) followed by Western blotting for incorporation of biotin-containing peptide into immunoprecipitated target protein(s) demonstrates that biotin-reactive bands in Western blots are cyclin A and CDK2 and that reactivity of these bands with benzo-CYS-9-48-bio peptide is dependent upon UV. (E) Cross-linking of benzophenone-containing peptides to increasing amounts of GST, cyclin A, or CDK2 demonstrates a strong preference of the peptide for cyclin A.

Techniques Used: Derivative Assay, Activity Assay, Irradiation, Binding Assay, Labeling, Immunoprecipitation, Western Blot

Addition of increasing amounts of GST-16E7 to CDK2 purified from insect cells or from bacteria results in a dose-dependent increase in kinase activity. Autoradiograph (A) demonstrates E7-dependent increase in phosphorylation of histone H1 by CDK2/cyclin A. Quantification of the experiment (B) plots PhosphorImager counts versus amount of GST-16E7 added. Results indicate that E7 affect peaks at 640 ng of E7 (or approximately 0.6 μM E7). CDK2/cyclin A reconstituted from bacterially expressed proteins is also activated by purified 16E7 (C).
Figure Legend Snippet: Addition of increasing amounts of GST-16E7 to CDK2 purified from insect cells or from bacteria results in a dose-dependent increase in kinase activity. Autoradiograph (A) demonstrates E7-dependent increase in phosphorylation of histone H1 by CDK2/cyclin A. Quantification of the experiment (B) plots PhosphorImager counts versus amount of GST-16E7 added. Results indicate that E7 affect peaks at 640 ng of E7 (or approximately 0.6 μM E7). CDK2/cyclin A reconstituted from bacterially expressed proteins is also activated by purified 16E7 (C).

Techniques Used: Purification, Activity Assay, Autoradiography

GST-16E7 activates purified cyclin E/CDK2 and cyclin A/CDK2 complexes. Autoradiographs demonstrate phosphorylation of histone H1 and Rb (pRb) by CDK2/cyclin E (A) and CDK2/cyclin A (B). (A) E7 potently upregulates CDK2/cyclin E histone H1 kinase activity while having modest effects on Rb kinase activity. Addition of p27 eliminates this effect and reduces activity to below baseline levels. (B) CDK2/cyclin A histone kinase activity is also dramatically upregulated by E7, and the effect is negated by addition of p27.
Figure Legend Snippet: GST-16E7 activates purified cyclin E/CDK2 and cyclin A/CDK2 complexes. Autoradiographs demonstrate phosphorylation of histone H1 and Rb (pRb) by CDK2/cyclin E (A) and CDK2/cyclin A (B). (A) E7 potently upregulates CDK2/cyclin E histone H1 kinase activity while having modest effects on Rb kinase activity. Addition of p27 eliminates this effect and reduces activity to below baseline levels. (B) CDK2/cyclin A histone kinase activity is also dramatically upregulated by E7, and the effect is negated by addition of p27.

Techniques Used: Purification, Activity Assay

16) Product Images from "Induction of G2M Arrest by Flavokawain A, a Kava Chalcone, Increases the Responsiveness of HER2-Overexpressing Breast Cancer Cells to Herceptin"

Article Title: Induction of G2M Arrest by Flavokawain A, a Kava Chalcone, Increases the Responsiveness of HER2-Overexpressing Breast Cancer Cells to Herceptin

Journal: Molecules (Basel, Switzerland)

doi: 10.3390/molecules22030462

FKA increases MPM-2 phosphorylation and Cdc kinase activity via inhibition of Cdc2 and Cdc25 phosphorylation and downregulation of Myt1 and Wee1 expression. ( A ) Cdc2 associated histone H1 kinase activity was decreased dose-dependently by FKA treatment for 24 h in SKBR3 cells; ( B – D ) SKBR3 cells were treated with indicated concentrations of FKA for 24 h. Phosphorylation levels of Cdc2, MPM-2, and Cdc25C as well as protein levels of cyclin B1, Myt1, and Wee1 were detected by Western blotting analysis. β-actin was detected as a loading control. A representative blot was shown from three independent experiments.
Figure Legend Snippet: FKA increases MPM-2 phosphorylation and Cdc kinase activity via inhibition of Cdc2 and Cdc25 phosphorylation and downregulation of Myt1 and Wee1 expression. ( A ) Cdc2 associated histone H1 kinase activity was decreased dose-dependently by FKA treatment for 24 h in SKBR3 cells; ( B – D ) SKBR3 cells were treated with indicated concentrations of FKA for 24 h. Phosphorylation levels of Cdc2, MPM-2, and Cdc25C as well as protein levels of cyclin B1, Myt1, and Wee1 were detected by Western blotting analysis. β-actin was detected as a loading control. A representative blot was shown from three independent experiments.

Techniques Used: Activity Assay, Inhibition, Expressing, Western Blot

17) Product Images from "p53 Regulation of G2 Checkpoint Is Retinoblastoma Protein Dependent"

Article Title: p53 Regulation of G2 Checkpoint Is Retinoblastoma Protein Dependent

Journal: Molecular and Cellular Biology

doi:

pRB is required for prolonged G 2 arrest and loss of cyclin B1 and Cdc2 protein levels. RKO-NEO and RKO-E7 cells were treated with IR (10 Gy) or ADR (350 nM), the drug was washed out at 17 h, and cells were harvested at 24, 48, and 72 h. (A and B) Cells were analyzed by flow cytometric analysis. (A) Propidium iodide fluorescence profile for control (Con) and treated cells; (B) quantifications from the flow histograms presented as percentages of cells with a 4 N DNA content after treatment with IR or ADR. Fifteen thousand events were analyzed for each condition, and all histograms were plotted by using the same scale for both axes. Protein lysates from cells exposed to IR (C) and ADR (D) were analyzed by Western blotting for the indicated proteins. (E) Protein lysates were analyzed for Cdc2 and Cdk2 kinase activities using histone H1 as a substrate. Results are representative of two independent experiments.
Figure Legend Snippet: pRB is required for prolonged G 2 arrest and loss of cyclin B1 and Cdc2 protein levels. RKO-NEO and RKO-E7 cells were treated with IR (10 Gy) or ADR (350 nM), the drug was washed out at 17 h, and cells were harvested at 24, 48, and 72 h. (A and B) Cells were analyzed by flow cytometric analysis. (A) Propidium iodide fluorescence profile for control (Con) and treated cells; (B) quantifications from the flow histograms presented as percentages of cells with a 4 N DNA content after treatment with IR or ADR. Fifteen thousand events were analyzed for each condition, and all histograms were plotted by using the same scale for both axes. Protein lysates from cells exposed to IR (C) and ADR (D) were analyzed by Western blotting for the indicated proteins. (E) Protein lysates were analyzed for Cdc2 and Cdk2 kinase activities using histone H1 as a substrate. Results are representative of two independent experiments.

Techniques Used: Flow Cytometry, Fluorescence, Western Blot

Dephosphorylation of pRB during p53 regulation of the G 2 checkpoint. (A) HIp53 cells were treated with 525 nM ADR for 17 h, the drug was washed out, and the cells were cultured for the indicated times in the presence or absence of PonA. (A) Cells were harvested and analyzed by Western blot analysis for pRB protein levels and phosphorylation state (upper blot) and for Cdk2 kinase activity using histone H1 as a substrate (lower blot). (B) HCT116, HCT116 p53 −/− , and HCT116 p21 −/− cells were treated with 350 nM ADR for the times indicated, and the cells were harvested and analyzed by Western blot analysis for pRB protein levels and phosphorylation state (upper blot) and for Cdk2 kinase activity using histone H1 as a substrate (lower blot). Results are representative of three independent experiments. C and Con, control.
Figure Legend Snippet: Dephosphorylation of pRB during p53 regulation of the G 2 checkpoint. (A) HIp53 cells were treated with 525 nM ADR for 17 h, the drug was washed out, and the cells were cultured for the indicated times in the presence or absence of PonA. (A) Cells were harvested and analyzed by Western blot analysis for pRB protein levels and phosphorylation state (upper blot) and for Cdk2 kinase activity using histone H1 as a substrate (lower blot). (B) HCT116, HCT116 p53 −/− , and HCT116 p21 −/− cells were treated with 350 nM ADR for the times indicated, and the cells were harvested and analyzed by Western blot analysis for pRB protein levels and phosphorylation state (upper blot) and for Cdk2 kinase activity using histone H1 as a substrate (lower blot). Results are representative of three independent experiments. C and Con, control.

Techniques Used: De-Phosphorylation Assay, Cell Culture, Western Blot, Activity Assay

p53 expression after IR or ADR inhibits cyclin B1-Cdc2 kinase activity. (A) Cells were treated with IR (8, 12, and 20 Gy) and incubated for 15 h, after which time cells were cultured for an additional 24, 48, or 72 h in the presence or absence of PonA. Protein lysates were analyzed by Western blotting for the indicated proteins. (B) Cells were treated with ADR (90, 175, 350, or 525 nM) for 17 h, the drug was washed out, and the cells were cultured an additional 24, 48, or 72 h in the presence or absence of PonA. (C and D) Protein lysates were analyzed by cyclin B1 immunoprecipitation-based assays for Cdc2 kinase activity using histone H1 as a substrate (C) and for immunoprecipitable cyclin B1, Cdc2, and p21 proteins by Western blotting (D). HIp53 cells were treated with 525 nM ADR for the data presented in panels C and D. Results are representative of two independent experiments. Con, control; IP, immunoprecipitation.
Figure Legend Snippet: p53 expression after IR or ADR inhibits cyclin B1-Cdc2 kinase activity. (A) Cells were treated with IR (8, 12, and 20 Gy) and incubated for 15 h, after which time cells were cultured for an additional 24, 48, or 72 h in the presence or absence of PonA. Protein lysates were analyzed by Western blotting for the indicated proteins. (B) Cells were treated with ADR (90, 175, 350, or 525 nM) for 17 h, the drug was washed out, and the cells were cultured an additional 24, 48, or 72 h in the presence or absence of PonA. (C and D) Protein lysates were analyzed by cyclin B1 immunoprecipitation-based assays for Cdc2 kinase activity using histone H1 as a substrate (C) and for immunoprecipitable cyclin B1, Cdc2, and p21 proteins by Western blotting (D). HIp53 cells were treated with 525 nM ADR for the data presented in panels C and D. Results are representative of two independent experiments. Con, control; IP, immunoprecipitation.

Techniques Used: Expressing, Activity Assay, Incubation, Cell Culture, Western Blot, Immunoprecipitation

18) Product Images from "Hepatocyte Growth Factor Releases Mink Epithelial Cells from Transforming Growth Factor ?1-Induced Growth Arrest by Restoring Cdk6 Expression and Cyclin E-Associated Cdk2 Activity"

Article Title: Hepatocyte Growth Factor Releases Mink Epithelial Cells from Transforming Growth Factor ?1-Induced Growth Arrest by Restoring Cdk6 Expression and Cyclin E-Associated Cdk2 Activity

Journal: Molecular and Cellular Biology

doi:

Cdk2- and cyclin E-associated kinase activities of TGF-β- and HGF-treated Mv1Lu cells. Cells were treated with the indicated growth factors, followed by lysis in NP-40 buffer. Immunoprecipitations with the indicated antibodies and kinase assays were carried out as described in Materials and Methods. (A) Cdk2-associated kinase activity with histone H1 as a substrate. (B) Cdk2 (lanes 1 to 4)- and cyclin E (lanes 5 to 8)-associated kinase activities with GST-Rb as a substrate. The figures show relative kinase activities compared to control cells (set at 100). (C) Sparsely seeded cells were pretreated with TGF-β (400 pM) for 16 h, followed by addition of HGF (220 pM) without removal of TGF-β, and incubations were continued for the indicated times (4 to 36 h). Untreated control cells are included (lanes 1 and 14). In wash , cells treated with TGF-β for 16 h were washed four times with growth medium, followed by addition of fresh medium. The cells were lysed, followed by Cdk6 and Cdk2 immunoblotting and determination of Cdk2-associated kinase activity towards histone H1. Relative kinase activities after subtraction of the kinase activity given by nonimmune serum are shown compared to cells treated with TGF-β for 16 h (set at 100) (lane 1). 5-BrdUrd incorporation was analyzed for the last hour of each incubation. The results are expressed as percent 5-BrdUrd-positive nuclei of Hoechst 33258-stained nuclei.
Figure Legend Snippet: Cdk2- and cyclin E-associated kinase activities of TGF-β- and HGF-treated Mv1Lu cells. Cells were treated with the indicated growth factors, followed by lysis in NP-40 buffer. Immunoprecipitations with the indicated antibodies and kinase assays were carried out as described in Materials and Methods. (A) Cdk2-associated kinase activity with histone H1 as a substrate. (B) Cdk2 (lanes 1 to 4)- and cyclin E (lanes 5 to 8)-associated kinase activities with GST-Rb as a substrate. The figures show relative kinase activities compared to control cells (set at 100). (C) Sparsely seeded cells were pretreated with TGF-β (400 pM) for 16 h, followed by addition of HGF (220 pM) without removal of TGF-β, and incubations were continued for the indicated times (4 to 36 h). Untreated control cells are included (lanes 1 and 14). In wash , cells treated with TGF-β for 16 h were washed four times with growth medium, followed by addition of fresh medium. The cells were lysed, followed by Cdk6 and Cdk2 immunoblotting and determination of Cdk2-associated kinase activity towards histone H1. Relative kinase activities after subtraction of the kinase activity given by nonimmune serum are shown compared to cells treated with TGF-β for 16 h (set at 100) (lane 1). 5-BrdUrd incorporation was analyzed for the last hour of each incubation. The results are expressed as percent 5-BrdUrd-positive nuclei of Hoechst 33258-stained nuclei.

Techniques Used: Lysis, Activity Assay, Incubation, Staining

(A) Growth factor regulation of cyclin E-associated kinase activity following p27 immunodepletion. Sparsely seeded cells were treated with TGF-β (100 pM) and HGF (220 pM), as indicated, followed by lysis in NP-40 buffer. Lysates were immunoprecipitated with cyclin E antibody (lanes 1 to 4) or with three subsequent cycles of polyclonal p27 antibody, followed by cyclin E immunoprecipitation (lanes 5 to 8). Kinase assays towards histone H1 were carried out as described in Materials and Methods. Relative kinase activities after subtraction of the kinase activity given by nonimmune serum compared to control cells (set at 100) are indicated. (B) Effects of p27 immunodepletion steps on cyclin E, Cdk2, p27, and their complexes. In parallel with experiment represented by panel A, cell lysates before and after three immunodepletion cycles with anti-p27 antibody were analyzed by immunoprecipitation (IP) with the indicated antibody, followed by immunoblotting (WB). Total cyclin E and p27 were analyzed by immunoblotting. Fold changes compared to controls are shown below each analysis.
Figure Legend Snippet: (A) Growth factor regulation of cyclin E-associated kinase activity following p27 immunodepletion. Sparsely seeded cells were treated with TGF-β (100 pM) and HGF (220 pM), as indicated, followed by lysis in NP-40 buffer. Lysates were immunoprecipitated with cyclin E antibody (lanes 1 to 4) or with three subsequent cycles of polyclonal p27 antibody, followed by cyclin E immunoprecipitation (lanes 5 to 8). Kinase assays towards histone H1 were carried out as described in Materials and Methods. Relative kinase activities after subtraction of the kinase activity given by nonimmune serum compared to control cells (set at 100) are indicated. (B) Effects of p27 immunodepletion steps on cyclin E, Cdk2, p27, and their complexes. In parallel with experiment represented by panel A, cell lysates before and after three immunodepletion cycles with anti-p27 antibody were analyzed by immunoprecipitation (IP) with the indicated antibody, followed by immunoblotting (WB). Total cyclin E and p27 were analyzed by immunoblotting. Fold changes compared to controls are shown below each analysis.

Techniques Used: Activity Assay, Lysis, Immunoprecipitation, Western Blot

19) Product Images from "Posttranslational Processing of Infected Cell Proteins 0 and 4 of Herpes Simplex Virus 1 Is Sequential and Reflects the Subcellular Compartment in Which the Proteins Localize"

Article Title: Posttranslational Processing of Infected Cell Proteins 0 and 4 of Herpes Simplex Virus 1 Is Sequential and Reflects the Subcellular Compartment in Which the Proteins Localize

Journal: Journal of Virology

doi: 10.1128/JVI.75.17.7904-7912.2001

Autoradiograms of histone H1 phosphorylated by in vitro kinase assays using immunoprecipitated cdk2 or cdc2 from HSV-1- or mock-infected cells. HEp-2 cells were HSV-1 or mock infected; 5 h after infection, the medium was replaced with medium containing 0, 10, or 100 μM roscovitine. Cells were harvested 12 h after infection, and cdk2 or cdc2 was immunoprecipitated (IP) with antibodie (Ab) as described in Materials and Methods. Kinase assays were done using histone H1 as the substrate. Phosphorylated histone H1 was quantitated by a PhosphorImager. Relative reduction in the phosphorylation of histone H1 due to roscovitine was determined relative to untreated samples.
Figure Legend Snippet: Autoradiograms of histone H1 phosphorylated by in vitro kinase assays using immunoprecipitated cdk2 or cdc2 from HSV-1- or mock-infected cells. HEp-2 cells were HSV-1 or mock infected; 5 h after infection, the medium was replaced with medium containing 0, 10, or 100 μM roscovitine. Cells were harvested 12 h after infection, and cdk2 or cdc2 was immunoprecipitated (IP) with antibodie (Ab) as described in Materials and Methods. Kinase assays were done using histone H1 as the substrate. Phosphorylated histone H1 was quantitated by a PhosphorImager. Relative reduction in the phosphorylation of histone H1 due to roscovitine was determined relative to untreated samples.

Techniques Used: In Vitro, Immunoprecipitation, Infection

20) Product Images from "Posttranslational Processing of Infected Cell Proteins 0 and 4 of Herpes Simplex Virus 1 Is Sequential and Reflects the Subcellular Compartment in Which the Proteins Localize"

Article Title: Posttranslational Processing of Infected Cell Proteins 0 and 4 of Herpes Simplex Virus 1 Is Sequential and Reflects the Subcellular Compartment in Which the Proteins Localize

Journal: Journal of Virology

doi: 10.1128/JVI.75.17.7904-7912.2001

Autoradiograms of histone H1 phosphorylated by in vitro kinase assays using immunoprecipitated cdk2 or cdc2 from HSV-1- or mock-infected cells. HEp-2 cells were HSV-1 or mock infected; 5 h after infection, the medium was replaced with medium containing 0, 10, or 100 μM roscovitine. Cells were harvested 12 h after infection, and cdk2 or cdc2 was immunoprecipitated (IP) with antibodie (Ab) as described in Materials and Methods. Kinase assays were done using histone H1 as the substrate. Phosphorylated histone H1 was quantitated by a PhosphorImager. Relative reduction in the phosphorylation of histone H1 due to roscovitine was determined relative to untreated samples.
Figure Legend Snippet: Autoradiograms of histone H1 phosphorylated by in vitro kinase assays using immunoprecipitated cdk2 or cdc2 from HSV-1- or mock-infected cells. HEp-2 cells were HSV-1 or mock infected; 5 h after infection, the medium was replaced with medium containing 0, 10, or 100 μM roscovitine. Cells were harvested 12 h after infection, and cdk2 or cdc2 was immunoprecipitated (IP) with antibodie (Ab) as described in Materials and Methods. Kinase assays were done using histone H1 as the substrate. Phosphorylated histone H1 was quantitated by a PhosphorImager. Relative reduction in the phosphorylation of histone H1 due to roscovitine was determined relative to untreated samples.

Techniques Used: In Vitro, Immunoprecipitation, Infection

21) Product Images from "NS1- and Minute Virus of Mice-Induced Cell Cycle Arrest: Involvement of p53 and p21cip1"

Article Title: NS1- and Minute Virus of Mice-Induced Cell Cycle Arrest: Involvement of p53 and p21cip1

Journal: Journal of Virology

doi: 10.1128/JVI.75.22.11071-11078.2001

NS1-induced p21 cip1 is associated with the cyclin A/Cdk and cyclin E/Cdk complexes and impairs cyclin A kinase activity. Exponentially growing cells were cultured in the presence (+) or absence (−) of dexamethasone for 24 h prior to harvesting. Proteins immunoprecipitated with anti-cyclin A (IPαA) (panels A and B) or anti-cyclin E (IPαE) (panel C) antibodies were analyzed for histone H1 kinase activity (A) and the presence of p21 cip1 by immunoblotting (B and C).
Figure Legend Snippet: NS1-induced p21 cip1 is associated with the cyclin A/Cdk and cyclin E/Cdk complexes and impairs cyclin A kinase activity. Exponentially growing cells were cultured in the presence (+) or absence (−) of dexamethasone for 24 h prior to harvesting. Proteins immunoprecipitated with anti-cyclin A (IPαA) (panels A and B) or anti-cyclin E (IPαE) (panel C) antibodies were analyzed for histone H1 kinase activity (A) and the presence of p21 cip1 by immunoblotting (B and C).

Techniques Used: Activity Assay, Cell Culture, Immunoprecipitation

22) Product Images from "Identification of a substrate-targeting domain in cyclin E necessary for phosphorylation of the retinoblastoma protein"

Article Title: Identification of a substrate-targeting domain in cyclin E necessary for phosphorylation of the retinoblastoma protein

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi:

Transfection of wild-type and mutant cyclin E proteins into SAOS-2 cells. ( A ) Percentage of cells in G 1 phase of the cell cycle versus amount of transfected plasmid. ( B ) SAOS-2 cells transfected with pCMVpRb plasmid and increasing amounts of cyclin E: percentage of cells in G 1 phase of the cell cycle versus amount of transfected plasmid. ( C ) Cyclin E-associated histone H1 kinase activity from cells transfected with pCMVpRb plus cyclin E ( D ) Phosphorylation of transfected full-length Rb by cyclin E (5 μg of transfected DNA) in SAOS-2 cells.
Figure Legend Snippet: Transfection of wild-type and mutant cyclin E proteins into SAOS-2 cells. ( A ) Percentage of cells in G 1 phase of the cell cycle versus amount of transfected plasmid. ( B ) SAOS-2 cells transfected with pCMVpRb plasmid and increasing amounts of cyclin E: percentage of cells in G 1 phase of the cell cycle versus amount of transfected plasmid. ( C ) Cyclin E-associated histone H1 kinase activity from cells transfected with pCMVpRb plus cyclin E ( D ) Phosphorylation of transfected full-length Rb by cyclin E (5 μg of transfected DNA) in SAOS-2 cells.

Techniques Used: Transfection, Mutagenesis, Plasmid Preparation, Activity Assay

VxCxE mutants are defective for Rb but not histone H1 phosphorylation in vitro . ( A ) Recombinant cyclin E–CDK2 complexes made in baculovirus-infected Sf-9 insect cells were incubated with [γ- 32 P]ATP and either histone H1 ( A ) or Rb ( B ) for increasing amounts of time. Results shown are representative of three different experiments.
Figure Legend Snippet: VxCxE mutants are defective for Rb but not histone H1 phosphorylation in vitro . ( A ) Recombinant cyclin E–CDK2 complexes made in baculovirus-infected Sf-9 insect cells were incubated with [γ- 32 P]ATP and either histone H1 ( A ) or Rb ( B ) for increasing amounts of time. Results shown are representative of three different experiments.

Techniques Used: In Vitro, Recombinant, Infection, Incubation

Transfection of wild-type and mutant cyclin E proteins into NIH 3T3 cells. NIH 3T3 cells were transiently transfected with increasing amounts of myc-tagged wild-type or mutant cyclin E. ( A ) Percentage of cells in G 1 phase of the cell cycle versus amount of transfected plasmid. ( B ) Cyclin E protein expression and associated histone H1 kinase activity from cells depicted in A.
Figure Legend Snippet: Transfection of wild-type and mutant cyclin E proteins into NIH 3T3 cells. NIH 3T3 cells were transiently transfected with increasing amounts of myc-tagged wild-type or mutant cyclin E. ( A ) Percentage of cells in G 1 phase of the cell cycle versus amount of transfected plasmid. ( B ) Cyclin E protein expression and associated histone H1 kinase activity from cells depicted in A.

Techniques Used: Transfection, Mutagenesis, Plasmid Preparation, Expressing, Activity Assay

23) Product Images from "c-Mos forces the mitotic cell cycle to undergo meiosis II to produce haploid gametes"

Article Title: c-Mos forces the mitotic cell cycle to undergo meiosis II to produce haploid gametes

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi:

Restoration of meiosis II by Mos in starfish oocytes in which translation of mos is prevented. ( A ) Formation of two polar bodies along with a female pronucleus after 1-MeAde addition to oocytes that had received the injection of antisense mos (AS) and either GST-Mos or ΔN-STE11. Polar bodies (arrowheads) were detected by DAPI staining. Black arrow indicates the female pronucleus. BrdUrd incorporation was undetectable in female pronuclei of GST-Mos or ΔN-STE11-restored oocytes but could be detected in control oocytes (white arrows). ( B ) Two limited rounds of H1 kinase activity after 1-MeAde addition to oocytes that had received the injection of antisense mos and the GST-Mos fusion protein. After 1-MeAde addition, oocytes were recovered at indicated times and processed for measurement of H1 kinase activity ( Top and Middle for autoradiogram; Bottom for radioactivity of the excised histone H1 bands; open circles, injection with antisense mos plus GST-Mos; closed squares, injection with antisense mos plus GST) and for Tyr phosphorylation in Cdc2 ( Bottom , Insets ). In both A and B , control oocytes had been injected with antisense mos (AS) and GST before 1-MeAde addition.
Figure Legend Snippet: Restoration of meiosis II by Mos in starfish oocytes in which translation of mos is prevented. ( A ) Formation of two polar bodies along with a female pronucleus after 1-MeAde addition to oocytes that had received the injection of antisense mos (AS) and either GST-Mos or ΔN-STE11. Polar bodies (arrowheads) were detected by DAPI staining. Black arrow indicates the female pronucleus. BrdUrd incorporation was undetectable in female pronuclei of GST-Mos or ΔN-STE11-restored oocytes but could be detected in control oocytes (white arrows). ( B ) Two limited rounds of H1 kinase activity after 1-MeAde addition to oocytes that had received the injection of antisense mos and the GST-Mos fusion protein. After 1-MeAde addition, oocytes were recovered at indicated times and processed for measurement of H1 kinase activity ( Top and Middle for autoradiogram; Bottom for radioactivity of the excised histone H1 bands; open circles, injection with antisense mos plus GST-Mos; closed squares, injection with antisense mos plus GST) and for Tyr phosphorylation in Cdc2 ( Bottom , Insets ). In both A and B , control oocytes had been injected with antisense mos (AS) and GST before 1-MeAde addition.

Techniques Used: Injection, Staining, Activity Assay, Radioactivity

24) Product Images from "Cell cycle inhibition by the anti-angiogenic agent TNP-470 is mediated by p53 and p21WAF1/CIP1"

Article Title: Cell cycle inhibition by the anti-angiogenic agent TNP-470 is mediated by p53 and p21WAF1/CIP1

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi:

Effect of TNP-470 on pRB phosphorylation, cyclin E-Cdk2 activity, and expression of cyclin E and Cdk2. HUVECs were synchronized by density arrest and replated at low density in the presence (+) or absence (−) of TNP-470 (10 nM), and harvested as described in Materials and Methods . ( A ) Western blot analysis of immunoprecipitated pRB. ( B ) In vitro kinase assay for cyclin E-dependent kinase activity. Samples were immunoprecipitated from lysates by using an anti-cyclin E Ab. Histone H1 was used as a substrate in the kinase reaction. ( C ) Western blot analysis of Cdk2, cyclin E, and actin expression levels.
Figure Legend Snippet: Effect of TNP-470 on pRB phosphorylation, cyclin E-Cdk2 activity, and expression of cyclin E and Cdk2. HUVECs were synchronized by density arrest and replated at low density in the presence (+) or absence (−) of TNP-470 (10 nM), and harvested as described in Materials and Methods . ( A ) Western blot analysis of immunoprecipitated pRB. ( B ) In vitro kinase assay for cyclin E-dependent kinase activity. Samples were immunoprecipitated from lysates by using an anti-cyclin E Ab. Histone H1 was used as a substrate in the kinase reaction. ( C ) Western blot analysis of Cdk2, cyclin E, and actin expression levels.

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

25) Product Images from "HIV-1 Vpr interacts with a human 34-kDa mov34 homologue, a cellular factor linked to the G2/M phase transition of the mammalian cell cycle"

Article Title: HIV-1 Vpr interacts with a human 34-kDa mov34 homologue, a cellular factor linked to the G2/M phase transition of the mammalian cell cycle

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi:

Effect of Vpr, hVIP/MOV34 sense, and hVIP/MOV34 antisense expression on histone H1 kinase activity. Cyclin B1 was immunoprecipitated from RD cell extracts and incubated with [γ- 32 P]ATP in the presence of exogenously added histone H1. Quantification of kinase activity by densitometry scanning was plotted. ( Inset ) Autoradiogram.
Figure Legend Snippet: Effect of Vpr, hVIP/MOV34 sense, and hVIP/MOV34 antisense expression on histone H1 kinase activity. Cyclin B1 was immunoprecipitated from RD cell extracts and incubated with [γ- 32 P]ATP in the presence of exogenously added histone H1. Quantification of kinase activity by densitometry scanning was plotted. ( Inset ) Autoradiogram.

Techniques Used: Expressing, Activity Assay, Immunoprecipitation, Incubation

26) Product Images from "Loss of G1/S Checkpoint in Human Immunodeficiency Virus Type 1-Infected Cells Is Associated with a Lack of Cyclin-Dependent Kinase Inhibitor p21/Waf1"

Article Title: Loss of G1/S Checkpoint in Human Immunodeficiency Virus Type 1-Infected Cells Is Associated with a Lack of Cyclin-Dependent Kinase Inhibitor p21/Waf1

Journal: Journal of Virology

doi:

Immunoprecipitation of cyclin E-associated complex in both infected and uninfected cells. Infected and uninfected cell extracts from gamma-irradiated cells were treated for immunoprecipitation with polyclonal rabbit anti-cyclin E antibody at 4°C. The immune complexes were pelleted the next day with protein A plus protein G beads, washed, and used for kinase assays with histone H1 or GST-Rb as substrates. (A) Labeled products resolved by SDS–4 to 20% PAGE and their corresponding counts with the Molecular Dynamics PhosphorImager software. (B) Straight Western blots for cdk2 and cyclin E in infected and uninfected cells. (C and D) Similar to panels A and B except that CEM (Vector) and CEM (Tat) were used as the starting materials for immunoprecipitations.
Figure Legend Snippet: Immunoprecipitation of cyclin E-associated complex in both infected and uninfected cells. Infected and uninfected cell extracts from gamma-irradiated cells were treated for immunoprecipitation with polyclonal rabbit anti-cyclin E antibody at 4°C. The immune complexes were pelleted the next day with protein A plus protein G beads, washed, and used for kinase assays with histone H1 or GST-Rb as substrates. (A) Labeled products resolved by SDS–4 to 20% PAGE and their corresponding counts with the Molecular Dynamics PhosphorImager software. (B) Straight Western blots for cdk2 and cyclin E in infected and uninfected cells. (C and D) Similar to panels A and B except that CEM (Vector) and CEM (Tat) were used as the starting materials for immunoprecipitations.

Techniques Used: Immunoprecipitation, Infection, Irradiation, Labeling, Polyacrylamide Gel Electrophoresis, Software, Western Blot, Plasmid Preparation

27) Product Images from "Mutant Caldesmon Lacking cdc2 Phosphorylation Sites Delays M-Phase Entry and Inhibits Cytokinesis"

Article Title: Mutant Caldesmon Lacking cdc2 Phosphorylation Sites Delays M-Phase Entry and Inhibits Cytokinesis

Journal: Molecular Biology of the Cell

doi:

No effects of 7th mutant caldesmon on cyclin B/cdc2 kinase activities. Histone H1 kinase activities of cyclin B1/cdc2 immune-complex were examined in the absence (control) or presence of wild-type or 7th mutant. The activities were expressed relative to the control. Four independent experiments were performed for each immune-complex.
Figure Legend Snippet: No effects of 7th mutant caldesmon on cyclin B/cdc2 kinase activities. Histone H1 kinase activities of cyclin B1/cdc2 immune-complex were examined in the absence (control) or presence of wild-type or 7th mutant. The activities were expressed relative to the control. Four independent experiments were performed for each immune-complex.

Techniques Used: Mutagenesis

28) Product Images from "Roughex Mediates G1 Arrest through a Physical Association with Cyclin A"

Article Title: Roughex Mediates G1 Arrest through a Physical Association with Cyclin A

Journal: Molecular and Cellular Biology

doi:

Rux inhibits CycA dependent kinase activity in cell extracts. The indicated volumes of extracts prepared from cells expressing wild-type (lanes 1 to 3 and 10 to 12) or mutant (lanes 4 to 9) forms of Rux were mixed with 2 μl of extract prepared from cells coexpressing CycA and Cdc2 (lanes 1 to 9) or CycA and Cdc2c (lanes 10 to 12) as described in Materials and Methods and tested for cyclin-CDK activity with histone H1 as an exogenous substrate. The reaction products were analyzed by SDS-PAGE and autoradiography. Wild-type and mutant Rux proteins were expressed at equivalent levels and migrated at the same relative size, as determined by Western blot analysis (data not shown). Increasing the volume of the Rux-containing extract from 0 to 3 μl resulted in an approximately fivefold inhibition of CycA-associated kinase activities as determined by PhosphorImager quantitation. When extract was prepared from untransfected or mocktransfected cells, a similar inhibition of endogenous CycA-CDK activity by Rux could be detected after prolonged exposure of autoradiographs (data not shown). Note that histone H1 phosphorylation products appear as a doublet in these experiments. The data are representative of at least three independent experiments.
Figure Legend Snippet: Rux inhibits CycA dependent kinase activity in cell extracts. The indicated volumes of extracts prepared from cells expressing wild-type (lanes 1 to 3 and 10 to 12) or mutant (lanes 4 to 9) forms of Rux were mixed with 2 μl of extract prepared from cells coexpressing CycA and Cdc2 (lanes 1 to 9) or CycA and Cdc2c (lanes 10 to 12) as described in Materials and Methods and tested for cyclin-CDK activity with histone H1 as an exogenous substrate. The reaction products were analyzed by SDS-PAGE and autoradiography. Wild-type and mutant Rux proteins were expressed at equivalent levels and migrated at the same relative size, as determined by Western blot analysis (data not shown). Increasing the volume of the Rux-containing extract from 0 to 3 μl resulted in an approximately fivefold inhibition of CycA-associated kinase activities as determined by PhosphorImager quantitation. When extract was prepared from untransfected or mocktransfected cells, a similar inhibition of endogenous CycA-CDK activity by Rux could be detected after prolonged exposure of autoradiographs (data not shown). Note that histone H1 phosphorylation products appear as a doublet in these experiments. The data are representative of at least three independent experiments.

Techniques Used: Activity Assay, Expressing, Mutagenesis, SDS Page, Autoradiography, Western Blot, Inhibition, Quantitation Assay

29) Product Images from "Assembly of cyclin D-dependent kinase and titration of p27Kip1 regulated by mitogen-activated protein kinase kinase (MEK1)"

Article Title: Assembly of cyclin D-dependent kinase and titration of p27Kip1 regulated by mitogen-activated protein kinase kinase (MEK1)

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi:

Overexpression of cyclin D1, CDK4, and MEK1* induces cyclin A synthesis and CDK2 activation, but not p27 Kip1 degradation. Cells (subclone 2) ectopically expressing cyclin D1, CDK4, and inducible MEK1* were starved and restimulated with ZnSO 4 or FBS. Cells harvested at intervals thereafter (hr) were assayed for cyclin A and p27 Kip1 expression by immunoblotting (top two panels), for cyclin D1-associated p27 Kip1 by immunoprecipitation with cyclin D1 followed by immunoblotting with anti-p27 Kip1 (middle panel), and for cyclin E- and CDK2-associated histone H1 kinase activity (bottom two panels). More than 85% of p27 Kip1 coprecipitated with cyclin D1.
Figure Legend Snippet: Overexpression of cyclin D1, CDK4, and MEK1* induces cyclin A synthesis and CDK2 activation, but not p27 Kip1 degradation. Cells (subclone 2) ectopically expressing cyclin D1, CDK4, and inducible MEK1* were starved and restimulated with ZnSO 4 or FBS. Cells harvested at intervals thereafter (hr) were assayed for cyclin A and p27 Kip1 expression by immunoblotting (top two panels), for cyclin D1-associated p27 Kip1 by immunoprecipitation with cyclin D1 followed by immunoblotting with anti-p27 Kip1 (middle panel), and for cyclin E- and CDK2-associated histone H1 kinase activity (bottom two panels). More than 85% of p27 Kip1 coprecipitated with cyclin D1.

Techniques Used: Over Expression, Activation Assay, Expressing, Immunoprecipitation, Activity Assay

Molecular correlates of S phase entry. ( A ) Cells from five individual subclones expressing inducible MEK1* were serum-starved and restimulated with ZnSO 4 or FBS for 16 hr, and the S phase fraction was determined by using flow cytometry to estimate the DNA content of propidium iodide-stained nuclei. ( B ) Serum-starved cells (subclone 3) stimulated with ZnSO 4 or FBS were assayed at 4-hr intervals for DNA content ( Upper ). Cyclin A, p21 Cip1 , and p27 Kip1 levels were determined by immunoblotting. Precipitates recovered with antibodies to cyclin E and CDK2 were assayed for histone H1 kinase activity.
Figure Legend Snippet: Molecular correlates of S phase entry. ( A ) Cells from five individual subclones expressing inducible MEK1* were serum-starved and restimulated with ZnSO 4 or FBS for 16 hr, and the S phase fraction was determined by using flow cytometry to estimate the DNA content of propidium iodide-stained nuclei. ( B ) Serum-starved cells (subclone 3) stimulated with ZnSO 4 or FBS were assayed at 4-hr intervals for DNA content ( Upper ). Cyclin A, p21 Cip1 , and p27 Kip1 levels were determined by immunoblotting. Precipitates recovered with antibodies to cyclin E and CDK2 were assayed for histone H1 kinase activity.

Techniques Used: Expressing, Flow Cytometry, Cytometry, Staining, Activity Assay

30) Product Images from "Identification of a substrate-targeting domain in cyclin E necessary for phosphorylation of the retinoblastoma protein"

Article Title: Identification of a substrate-targeting domain in cyclin E necessary for phosphorylation of the retinoblastoma protein

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi:

Transfection of wild-type and mutant cyclin E proteins into SAOS-2 cells. ( A ) Percentage of cells in G 1 phase of the cell cycle versus amount of transfected plasmid. ( B ) SAOS-2 cells transfected with pCMVpRb plasmid and increasing amounts of cyclin E: percentage of cells in G 1 phase of the cell cycle versus amount of transfected plasmid. ( C ) Cyclin E-associated histone H1 kinase activity from cells transfected with pCMVpRb plus cyclin E ( D ) Phosphorylation of transfected full-length Rb by cyclin E (5 μg of transfected DNA) in SAOS-2 cells.
Figure Legend Snippet: Transfection of wild-type and mutant cyclin E proteins into SAOS-2 cells. ( A ) Percentage of cells in G 1 phase of the cell cycle versus amount of transfected plasmid. ( B ) SAOS-2 cells transfected with pCMVpRb plasmid and increasing amounts of cyclin E: percentage of cells in G 1 phase of the cell cycle versus amount of transfected plasmid. ( C ) Cyclin E-associated histone H1 kinase activity from cells transfected with pCMVpRb plus cyclin E ( D ) Phosphorylation of transfected full-length Rb by cyclin E (5 μg of transfected DNA) in SAOS-2 cells.

Techniques Used: Transfection, Mutagenesis, Plasmid Preparation, Activity Assay

VxCxE mutants are defective for Rb but not histone H1 phosphorylation in vitro . ( A ) Recombinant cyclin E–CDK2 complexes made in baculovirus-infected Sf-9 insect cells were incubated with [γ- 32 P]ATP and either histone H1 ( A ) or Rb ( B ) for increasing amounts of time. Results shown are representative of three different experiments.
Figure Legend Snippet: VxCxE mutants are defective for Rb but not histone H1 phosphorylation in vitro . ( A ) Recombinant cyclin E–CDK2 complexes made in baculovirus-infected Sf-9 insect cells were incubated with [γ- 32 P]ATP and either histone H1 ( A ) or Rb ( B ) for increasing amounts of time. Results shown are representative of three different experiments.

Techniques Used: In Vitro, Recombinant, Infection, Incubation

Transfection of wild-type and mutant cyclin E proteins into NIH 3T3 cells. NIH 3T3 cells were transiently transfected with increasing amounts of myc-tagged wild-type or mutant cyclin E. ( A ) Percentage of cells in G 1 phase of the cell cycle versus amount of transfected plasmid. ( B ) Cyclin E protein expression and associated histone H1 kinase activity from cells depicted in A.
Figure Legend Snippet: Transfection of wild-type and mutant cyclin E proteins into NIH 3T3 cells. NIH 3T3 cells were transiently transfected with increasing amounts of myc-tagged wild-type or mutant cyclin E. ( A ) Percentage of cells in G 1 phase of the cell cycle versus amount of transfected plasmid. ( B ) Cyclin E protein expression and associated histone H1 kinase activity from cells depicted in A.

Techniques Used: Transfection, Mutagenesis, Plasmid Preparation, Expressing, Activity Assay

31) Product Images from "Direct Activation of Cyclin-Dependent Kinase 2 by Human Papillomavirus E7"

Article Title: Direct Activation of Cyclin-Dependent Kinase 2 by Human Papillomavirus E7

Journal: Journal of Virology

doi: 10.1128/JVI.77.19.10566-10574.2003

Cross-linking of E7 peptides to CDK2/cyclin A complex. (A) HPV16 E7 (1.8 μM) and E7-derived peptides (50 μM) stimulate CDK2/cyclin A histone kinase activity. (B) Benzo-CYS-9-48-bio and benzo-NH 2 -9-48-bio peptides associate with cyclin A/CDK2 complex following UV irradiation. Histone H1 is not cross-linked under identical conditions. (C) CDK2/cyclin A (0.1 μM) and benzo-CYS-9-48-bio (0.5 μM) were with or without competitor E7 or E7 peptide. Both full-length 16E7 and the 9-48 16E7 peptide compete with the benzo-CYS-9-48-bio peptide for binding to the cyclin A/CDK2 complex. These results suggest that labeling of the CDK2/cyclin A complex by benzo-CYS-9-48-bio is specific and is not due to indiscriminate labeling of the complex. (D) Immunoprecipitation (IP) followed by Western blotting for incorporation of biotin-containing peptide into immunoprecipitated target protein(s) demonstrates that biotin-reactive bands in Western blots are cyclin A and CDK2 and that reactivity of these bands with benzo-CYS-9-48-bio peptide is dependent upon UV. (E) Cross-linking of benzophenone-containing peptides to increasing amounts of GST, cyclin A, or CDK2 demonstrates a strong preference of the peptide for cyclin A.
Figure Legend Snippet: Cross-linking of E7 peptides to CDK2/cyclin A complex. (A) HPV16 E7 (1.8 μM) and E7-derived peptides (50 μM) stimulate CDK2/cyclin A histone kinase activity. (B) Benzo-CYS-9-48-bio and benzo-NH 2 -9-48-bio peptides associate with cyclin A/CDK2 complex following UV irradiation. Histone H1 is not cross-linked under identical conditions. (C) CDK2/cyclin A (0.1 μM) and benzo-CYS-9-48-bio (0.5 μM) were with or without competitor E7 or E7 peptide. Both full-length 16E7 and the 9-48 16E7 peptide compete with the benzo-CYS-9-48-bio peptide for binding to the cyclin A/CDK2 complex. These results suggest that labeling of the CDK2/cyclin A complex by benzo-CYS-9-48-bio is specific and is not due to indiscriminate labeling of the complex. (D) Immunoprecipitation (IP) followed by Western blotting for incorporation of biotin-containing peptide into immunoprecipitated target protein(s) demonstrates that biotin-reactive bands in Western blots are cyclin A and CDK2 and that reactivity of these bands with benzo-CYS-9-48-bio peptide is dependent upon UV. (E) Cross-linking of benzophenone-containing peptides to increasing amounts of GST, cyclin A, or CDK2 demonstrates a strong preference of the peptide for cyclin A.

Techniques Used: Derivative Assay, Activity Assay, Irradiation, Binding Assay, Labeling, Immunoprecipitation, Western Blot

Addition of increasing amounts of GST-16E7 to CDK2 purified from insect cells or from bacteria results in a dose-dependent increase in kinase activity. Autoradiograph (A) demonstrates E7-dependent increase in phosphorylation of histone H1 by CDK2/cyclin A. Quantification of the experiment (B) plots PhosphorImager counts versus amount of GST-16E7 added. Results indicate that E7 affect peaks at 640 ng of E7 (or approximately 0.6 μM E7). CDK2/cyclin A reconstituted from bacterially expressed proteins is also activated by purified 16E7 (C).
Figure Legend Snippet: Addition of increasing amounts of GST-16E7 to CDK2 purified from insect cells or from bacteria results in a dose-dependent increase in kinase activity. Autoradiograph (A) demonstrates E7-dependent increase in phosphorylation of histone H1 by CDK2/cyclin A. Quantification of the experiment (B) plots PhosphorImager counts versus amount of GST-16E7 added. Results indicate that E7 affect peaks at 640 ng of E7 (or approximately 0.6 μM E7). CDK2/cyclin A reconstituted from bacterially expressed proteins is also activated by purified 16E7 (C).

Techniques Used: Purification, Activity Assay, Autoradiography

GST-16E7 activates purified cyclin E/CDK2 and cyclin A/CDK2 complexes. Autoradiographs demonstrate phosphorylation of histone H1 and Rb (pRb) by CDK2/cyclin E (A) and CDK2/cyclin A (B). (A) E7 potently upregulates CDK2/cyclin E histone H1 kinase activity while having modest effects on Rb kinase activity. Addition of p27 eliminates this effect and reduces activity to below baseline levels. (B) CDK2/cyclin A histone kinase activity is also dramatically upregulated by E7, and the effect is negated by addition of p27.
Figure Legend Snippet: GST-16E7 activates purified cyclin E/CDK2 and cyclin A/CDK2 complexes. Autoradiographs demonstrate phosphorylation of histone H1 and Rb (pRb) by CDK2/cyclin E (A) and CDK2/cyclin A (B). (A) E7 potently upregulates CDK2/cyclin E histone H1 kinase activity while having modest effects on Rb kinase activity. Addition of p27 eliminates this effect and reduces activity to below baseline levels. (B) CDK2/cyclin A histone kinase activity is also dramatically upregulated by E7, and the effect is negated by addition of p27.

Techniques Used: Purification, Activity Assay

32) Product Images from "Direct Activation of Cyclin-Dependent Kinase 2 by Human Papillomavirus E7"

Article Title: Direct Activation of Cyclin-Dependent Kinase 2 by Human Papillomavirus E7

Journal: Journal of Virology

doi: 10.1128/JVI.77.19.10566-10574.2003

Cross-linking of E7 peptides to CDK2/cyclin A complex. (A) HPV16 E7 (1.8 μM) and E7-derived peptides (50 μM) stimulate CDK2/cyclin A histone kinase activity. (B) Benzo-CYS-9-48-bio and benzo-NH 2 -9-48-bio peptides associate with cyclin A/CDK2 complex following UV irradiation. Histone H1 is not cross-linked under identical conditions. (C) CDK2/cyclin A (0.1 μM) and benzo-CYS-9-48-bio (0.5 μM) were with or without competitor E7 or E7 peptide. Both full-length 16E7 and the 9-48 16E7 peptide compete with the benzo-CYS-9-48-bio peptide for binding to the cyclin A/CDK2 complex. These results suggest that labeling of the CDK2/cyclin A complex by benzo-CYS-9-48-bio is specific and is not due to indiscriminate labeling of the complex. (D) Immunoprecipitation (IP) followed by Western blotting for incorporation of biotin-containing peptide into immunoprecipitated target protein(s) demonstrates that biotin-reactive bands in Western blots are cyclin A and CDK2 and that reactivity of these bands with benzo-CYS-9-48-bio peptide is dependent upon UV. (E) Cross-linking of benzophenone-containing peptides to increasing amounts of GST, cyclin A, or CDK2 demonstrates a strong preference of the peptide for cyclin A.
Figure Legend Snippet: Cross-linking of E7 peptides to CDK2/cyclin A complex. (A) HPV16 E7 (1.8 μM) and E7-derived peptides (50 μM) stimulate CDK2/cyclin A histone kinase activity. (B) Benzo-CYS-9-48-bio and benzo-NH 2 -9-48-bio peptides associate with cyclin A/CDK2 complex following UV irradiation. Histone H1 is not cross-linked under identical conditions. (C) CDK2/cyclin A (0.1 μM) and benzo-CYS-9-48-bio (0.5 μM) were with or without competitor E7 or E7 peptide. Both full-length 16E7 and the 9-48 16E7 peptide compete with the benzo-CYS-9-48-bio peptide for binding to the cyclin A/CDK2 complex. These results suggest that labeling of the CDK2/cyclin A complex by benzo-CYS-9-48-bio is specific and is not due to indiscriminate labeling of the complex. (D) Immunoprecipitation (IP) followed by Western blotting for incorporation of biotin-containing peptide into immunoprecipitated target protein(s) demonstrates that biotin-reactive bands in Western blots are cyclin A and CDK2 and that reactivity of these bands with benzo-CYS-9-48-bio peptide is dependent upon UV. (E) Cross-linking of benzophenone-containing peptides to increasing amounts of GST, cyclin A, or CDK2 demonstrates a strong preference of the peptide for cyclin A.

Techniques Used: Derivative Assay, Activity Assay, Irradiation, Binding Assay, Labeling, Immunoprecipitation, Western Blot

Addition of increasing amounts of GST-16E7 to CDK2 purified from insect cells or from bacteria results in a dose-dependent increase in kinase activity. Autoradiograph (A) demonstrates E7-dependent increase in phosphorylation of histone H1 by CDK2/cyclin A. Quantification of the experiment (B) plots PhosphorImager counts versus amount of GST-16E7 added. Results indicate that E7 affect peaks at 640 ng of E7 (or approximately 0.6 μM E7). CDK2/cyclin A reconstituted from bacterially expressed proteins is also activated by purified 16E7 (C).
Figure Legend Snippet: Addition of increasing amounts of GST-16E7 to CDK2 purified from insect cells or from bacteria results in a dose-dependent increase in kinase activity. Autoradiograph (A) demonstrates E7-dependent increase in phosphorylation of histone H1 by CDK2/cyclin A. Quantification of the experiment (B) plots PhosphorImager counts versus amount of GST-16E7 added. Results indicate that E7 affect peaks at 640 ng of E7 (or approximately 0.6 μM E7). CDK2/cyclin A reconstituted from bacterially expressed proteins is also activated by purified 16E7 (C).

Techniques Used: Purification, Activity Assay, Autoradiography

GST-16E7 activates purified cyclin E/CDK2 and cyclin A/CDK2 complexes. Autoradiographs demonstrate phosphorylation of histone H1 and Rb (pRb) by CDK2/cyclin E (A) and CDK2/cyclin A (B). (A) E7 potently upregulates CDK2/cyclin E histone H1 kinase activity while having modest effects on Rb kinase activity. Addition of p27 eliminates this effect and reduces activity to below baseline levels. (B) CDK2/cyclin A histone kinase activity is also dramatically upregulated by E7, and the effect is negated by addition of p27.
Figure Legend Snippet: GST-16E7 activates purified cyclin E/CDK2 and cyclin A/CDK2 complexes. Autoradiographs demonstrate phosphorylation of histone H1 and Rb (pRb) by CDK2/cyclin E (A) and CDK2/cyclin A (B). (A) E7 potently upregulates CDK2/cyclin E histone H1 kinase activity while having modest effects on Rb kinase activity. Addition of p27 eliminates this effect and reduces activity to below baseline levels. (B) CDK2/cyclin A histone kinase activity is also dramatically upregulated by E7, and the effect is negated by addition of p27.

Techniques Used: Purification, Activity Assay

33) Product Images from "Aberrant Expression of Mitotic Cdc2/Cyclin B1 Kinase in Degenerating Neurons of Alzheimer’s Disease Brain"

Article Title: Aberrant Expression of Mitotic Cdc2/Cyclin B1 Kinase in Degenerating Neurons of Alzheimer’s Disease Brain

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.17-10-03588.1997

Preferential creation of the TG/MC phospho-epitopes in recombinant tau by cdc2/cyclin B. A , 32 P incorporation. Histone H1 and tau were incubated with the indicated proline-directed kinases. Bottom panels (i.e., gel) for each substrate show Coomassie blue staining of the protein in the gel, and the top panels (i.e., autorad) show the corresponding autoradiograms. Equivalent amounts of 32 P were incorporated into H1 after incubation with the indicated kinases. This was also the case with tau as substrate, except that higher amounts of 32 P incorporation were observed with MAPK. The positions of molecular weight (MW) markers in kilodaltons are shown on the left of each panel. B , Tau immunoreactivity. Replicate panels of tau protein phosphorylated with MAPK, cdc2/cyclin B, cdk5, or GSK-3, respectively, were stained with the TG/MC and PHF-1 antibodies as indicated on the left of each panel. Whereas all the kinases produced the PHF-1 phospho-epitope in tau, only cdc2/cyclin B produced the mitotic TG/MC epitopes. The position of the 70 kDa MW marker is shown on the left of each panel.
Figure Legend Snippet: Preferential creation of the TG/MC phospho-epitopes in recombinant tau by cdc2/cyclin B. A , 32 P incorporation. Histone H1 and tau were incubated with the indicated proline-directed kinases. Bottom panels (i.e., gel) for each substrate show Coomassie blue staining of the protein in the gel, and the top panels (i.e., autorad) show the corresponding autoradiograms. Equivalent amounts of 32 P were incorporated into H1 after incubation with the indicated kinases. This was also the case with tau as substrate, except that higher amounts of 32 P incorporation were observed with MAPK. The positions of molecular weight (MW) markers in kilodaltons are shown on the left of each panel. B , Tau immunoreactivity. Replicate panels of tau protein phosphorylated with MAPK, cdc2/cyclin B, cdk5, or GSK-3, respectively, were stained with the TG/MC and PHF-1 antibodies as indicated on the left of each panel. Whereas all the kinases produced the PHF-1 phospho-epitope in tau, only cdc2/cyclin B produced the mitotic TG/MC epitopes. The position of the 70 kDa MW marker is shown on the left of each panel.

Techniques Used: Recombinant, Incubation, Staining, Molecular Weight, Produced, Marker

Mitotic kinase activity is higher in AD than in normal brain. Normal and AD extracts were subjected to precipitation with p13suc1-agarose and the precipitates assayed for histone H-1 phosphorylation activity. Representative data from two sets of four normal and four AD cases are shown. Top panels show the histone H1 bands and, with the hippocampus, the position of the GST-p13suc1 fusion product as revealed by Coomassie blue staining. P13suc1-agarose and GST-p13suc1-agarose gave identical results and were used interchangeably. Bottom panels show the corresponding autoradiograms. Phosphorylation of H1 measured by Phosphoimager is higher in AD than in normal brain.
Figure Legend Snippet: Mitotic kinase activity is higher in AD than in normal brain. Normal and AD extracts were subjected to precipitation with p13suc1-agarose and the precipitates assayed for histone H-1 phosphorylation activity. Representative data from two sets of four normal and four AD cases are shown. Top panels show the histone H1 bands and, with the hippocampus, the position of the GST-p13suc1 fusion product as revealed by Coomassie blue staining. P13suc1-agarose and GST-p13suc1-agarose gave identical results and were used interchangeably. Bottom panels show the corresponding autoradiograms. Phosphorylation of H1 measured by Phosphoimager is higher in AD than in normal brain.

Techniques Used: Activity Assay, Staining

34) Product Images from "Direct Activation of Cyclin-Dependent Kinase 2 by Human Papillomavirus E7"

Article Title: Direct Activation of Cyclin-Dependent Kinase 2 by Human Papillomavirus E7

Journal: Journal of Virology

doi: 10.1128/JVI.77.19.10566-10574.2003

Cross-linking of E7 peptides to CDK2/cyclin A complex. (A) HPV16 E7 (1.8 μM) and E7-derived peptides (50 μM) stimulate CDK2/cyclin A histone kinase activity. (B) Benzo-CYS-9-48-bio and benzo-NH 2 -9-48-bio peptides associate with cyclin A/CDK2 complex following UV irradiation. Histone H1 is not cross-linked under identical conditions. (C) CDK2/cyclin A (0.1 μM) and benzo-CYS-9-48-bio (0.5 μM) were with or without competitor E7 or E7 peptide. Both full-length 16E7 and the 9-48 16E7 peptide compete with the benzo-CYS-9-48-bio peptide for binding to the cyclin A/CDK2 complex. These results suggest that labeling of the CDK2/cyclin A complex by benzo-CYS-9-48-bio is specific and is not due to indiscriminate labeling of the complex. (D) Immunoprecipitation (IP) followed by Western blotting for incorporation of biotin-containing peptide into immunoprecipitated target protein(s) demonstrates that biotin-reactive bands in Western blots are cyclin A and CDK2 and that reactivity of these bands with benzo-CYS-9-48-bio peptide is dependent upon UV. (E) Cross-linking of benzophenone-containing peptides to increasing amounts of GST, cyclin A, or CDK2 demonstrates a strong preference of the peptide for cyclin A.
Figure Legend Snippet: Cross-linking of E7 peptides to CDK2/cyclin A complex. (A) HPV16 E7 (1.8 μM) and E7-derived peptides (50 μM) stimulate CDK2/cyclin A histone kinase activity. (B) Benzo-CYS-9-48-bio and benzo-NH 2 -9-48-bio peptides associate with cyclin A/CDK2 complex following UV irradiation. Histone H1 is not cross-linked under identical conditions. (C) CDK2/cyclin A (0.1 μM) and benzo-CYS-9-48-bio (0.5 μM) were with or without competitor E7 or E7 peptide. Both full-length 16E7 and the 9-48 16E7 peptide compete with the benzo-CYS-9-48-bio peptide for binding to the cyclin A/CDK2 complex. These results suggest that labeling of the CDK2/cyclin A complex by benzo-CYS-9-48-bio is specific and is not due to indiscriminate labeling of the complex. (D) Immunoprecipitation (IP) followed by Western blotting for incorporation of biotin-containing peptide into immunoprecipitated target protein(s) demonstrates that biotin-reactive bands in Western blots are cyclin A and CDK2 and that reactivity of these bands with benzo-CYS-9-48-bio peptide is dependent upon UV. (E) Cross-linking of benzophenone-containing peptides to increasing amounts of GST, cyclin A, or CDK2 demonstrates a strong preference of the peptide for cyclin A.

Techniques Used: Derivative Assay, Activity Assay, Irradiation, Binding Assay, Labeling, Immunoprecipitation, Western Blot

Addition of increasing amounts of GST-16E7 to CDK2 purified from insect cells or from bacteria results in a dose-dependent increase in kinase activity. Autoradiograph (A) demonstrates E7-dependent increase in phosphorylation of histone H1 by CDK2/cyclin A. Quantification of the experiment (B) plots PhosphorImager counts versus amount of GST-16E7 added. Results indicate that E7 affect peaks at 640 ng of E7 (or approximately 0.6 μM E7). CDK2/cyclin A reconstituted from bacterially expressed proteins is also activated by purified 16E7 (C).
Figure Legend Snippet: Addition of increasing amounts of GST-16E7 to CDK2 purified from insect cells or from bacteria results in a dose-dependent increase in kinase activity. Autoradiograph (A) demonstrates E7-dependent increase in phosphorylation of histone H1 by CDK2/cyclin A. Quantification of the experiment (B) plots PhosphorImager counts versus amount of GST-16E7 added. Results indicate that E7 affect peaks at 640 ng of E7 (or approximately 0.6 μM E7). CDK2/cyclin A reconstituted from bacterially expressed proteins is also activated by purified 16E7 (C).

Techniques Used: Purification, Activity Assay, Autoradiography

GST-16E7 activates purified cyclin E/CDK2 and cyclin A/CDK2 complexes. Autoradiographs demonstrate phosphorylation of histone H1 and Rb (pRb) by CDK2/cyclin E (A) and CDK2/cyclin A (B). (A) E7 potently upregulates CDK2/cyclin E histone H1 kinase activity while having modest effects on Rb kinase activity. Addition of p27 eliminates this effect and reduces activity to below baseline levels. (B) CDK2/cyclin A histone kinase activity is also dramatically upregulated by E7, and the effect is negated by addition of p27.
Figure Legend Snippet: GST-16E7 activates purified cyclin E/CDK2 and cyclin A/CDK2 complexes. Autoradiographs demonstrate phosphorylation of histone H1 and Rb (pRb) by CDK2/cyclin E (A) and CDK2/cyclin A (B). (A) E7 potently upregulates CDK2/cyclin E histone H1 kinase activity while having modest effects on Rb kinase activity. Addition of p27 eliminates this effect and reduces activity to below baseline levels. (B) CDK2/cyclin A histone kinase activity is also dramatically upregulated by E7, and the effect is negated by addition of p27.

Techniques Used: Purification, Activity Assay

35) Product Images from "Inositol Hexaphosphate Inhibits Growth and Induces G1 Arrest and Apoptotic Death of Androgen-Dependent Human Prostate Carcinoma LNCaP Cells 1"

Article Title: Inositol Hexaphosphate Inhibits Growth and Induces G1 Arrest and Apoptotic Death of Androgen-Dependent Human Prostate Carcinoma LNCaP Cells 1

Journal: Neoplasia (New York, N.Y.)

doi:

Effect of IP6 on CDKI-CDK binding and CDK kinase activity in LNCaP cells. Cells at 60% confluency under standard culture conditions were treated with different doses of IP6 for 12 or 24 hours, and cell lysates were prepared in nondenaturing lysis buffer as detailed in Materials and Methods section. For CDKI-CDK bindings (A), Cip1/p21 or Kip1/p27 was immunoprecipitated from the total cell lysates and subjected to SDS-PAGE followed by immunoblotting. The membranes in both cases were probed with anti-CDK2 and anti-CDK4 antibodies followed by peroxidase-conjugated appropriate secondary antibody and visualization by ECL detection system. For CDK2 kinase activity (B), CDK2 was immunoprecipitated from the total cell lysates and subjected to kinase assay in the presence of [γ- 32 P] ATP and histone H1 as substrate, as detailed in Materials and Methods section. Samples were then subjected to SDS-PAGE followed by gel drying and autoradiography. For CDK4 kinase activity (C), CDK4 was immunoprecipitated from the total cell lysates and subjected to kinase assay in the presence of ATP and Rb fusion protein as substrate, as detailed in Materials and Methods section. Samples were then subjected to SDS-PAGE followed by immunoblotting. The membranes were probed with phosphoserine-specific Rb antibodies followed by peroxidase-conjugated appropriate secondary antibody and visualization by ECL detection system. Different treatments are as labeled in the figure.
Figure Legend Snippet: Effect of IP6 on CDKI-CDK binding and CDK kinase activity in LNCaP cells. Cells at 60% confluency under standard culture conditions were treated with different doses of IP6 for 12 or 24 hours, and cell lysates were prepared in nondenaturing lysis buffer as detailed in Materials and Methods section. For CDKI-CDK bindings (A), Cip1/p21 or Kip1/p27 was immunoprecipitated from the total cell lysates and subjected to SDS-PAGE followed by immunoblotting. The membranes in both cases were probed with anti-CDK2 and anti-CDK4 antibodies followed by peroxidase-conjugated appropriate secondary antibody and visualization by ECL detection system. For CDK2 kinase activity (B), CDK2 was immunoprecipitated from the total cell lysates and subjected to kinase assay in the presence of [γ- 32 P] ATP and histone H1 as substrate, as detailed in Materials and Methods section. Samples were then subjected to SDS-PAGE followed by gel drying and autoradiography. For CDK4 kinase activity (C), CDK4 was immunoprecipitated from the total cell lysates and subjected to kinase assay in the presence of ATP and Rb fusion protein as substrate, as detailed in Materials and Methods section. Samples were then subjected to SDS-PAGE followed by immunoblotting. The membranes were probed with phosphoserine-specific Rb antibodies followed by peroxidase-conjugated appropriate secondary antibody and visualization by ECL detection system. Different treatments are as labeled in the figure.

Techniques Used: Binding Assay, Activity Assay, Lysis, Immunoprecipitation, SDS Page, Kinase Assay, Autoradiography, Labeling

36) Product Images from "Nuclear Localization of Cyclin B1 Controls Mitotic Entry After DNA Damage "

Article Title: Nuclear Localization of Cyclin B1 Controls Mitotic Entry After DNA Damage

Journal: The Journal of Cell Biology

doi:

Effects of nuclear cyclin B1 and Cdc2AF on progression through mitosis. ( A ) HeLa cells were synchronized at the G1/S boundary by a double thymidine treatment and infected for 3 h with recombinant adenoviruses encoding the tTA transactivator ( a and f , control) or with multiple viruses encoding tTA plus cyclin B1 ( b and g , B1), NLS-cyclin B1 ( c and h , NB1), both cyclin B1 and Cdc2AF ( d and i , B1 + AF), or both NLS-cyclin B1 and Cdc2AF ( e and j ; NB1 + AF). 4 h after release from G1/S arrest, cells were subjected to secondary immunofluorescence analysis with an antibody against the Myc epitope tag on cyclin B1 (α-Myc; a–e ) and treated with Hoechst 33258 to label nuclear DNA ( f–j ). ( B ) HeLa cells were synchronized at the G1/S boundary and infected for 4 h with recombinant adenoviruses encoding the tTA transactivator ( a , Control) or with multiple viruses encoding tTA plus cyclin B1 ( b , B1), NLS-cyclin B1 ( c , N.B1), Cdc2AF ( d , AF), both cyclin B1 and Cdc2AF ( e , B1 + AF), or both NLS-cyclin B1 and Cdc2AF ( f , N.B1 + AF). Cell lysates prepared at the indicated times after G1/S release were subjected to immunoblotting with antibodies against cyclin B1 ( left ), or antibodies against the PSTAIRE sequence conserved among CDKs ( middle ; Cdc2 is the major anti-PSTAIRE antigen in HeLa cells). Arrowheads indicate the epitope-tagged, virally-encoded cyclin B1, NLS-cyclin B1, and Cdc2AF proteins. Histone H1 kinase activity was measured in immunoprecipitates with anti-cyclin B1 ( right ); autoradiographic exposure times were 1.5 h. ( C ) Cells from the same experiment as in B were harvested at the indicated times, fixed, stained with propidium iodide, and analyzed by flow cytometry to measure DNA content. Abbreviations are as given in B . ( D ) A fraction of the cells prepared for flow cytometric analysis in C were examined by microscopy for the presence of condensed chromosomes. At least 300 cells were analyzed for each sample. These values represent the means of data obtained from two separate experiments, in which results were essentially identical. Abbreviations are as given in B .
Figure Legend Snippet: Effects of nuclear cyclin B1 and Cdc2AF on progression through mitosis. ( A ) HeLa cells were synchronized at the G1/S boundary by a double thymidine treatment and infected for 3 h with recombinant adenoviruses encoding the tTA transactivator ( a and f , control) or with multiple viruses encoding tTA plus cyclin B1 ( b and g , B1), NLS-cyclin B1 ( c and h , NB1), both cyclin B1 and Cdc2AF ( d and i , B1 + AF), or both NLS-cyclin B1 and Cdc2AF ( e and j ; NB1 + AF). 4 h after release from G1/S arrest, cells were subjected to secondary immunofluorescence analysis with an antibody against the Myc epitope tag on cyclin B1 (α-Myc; a–e ) and treated with Hoechst 33258 to label nuclear DNA ( f–j ). ( B ) HeLa cells were synchronized at the G1/S boundary and infected for 4 h with recombinant adenoviruses encoding the tTA transactivator ( a , Control) or with multiple viruses encoding tTA plus cyclin B1 ( b , B1), NLS-cyclin B1 ( c , N.B1), Cdc2AF ( d , AF), both cyclin B1 and Cdc2AF ( e , B1 + AF), or both NLS-cyclin B1 and Cdc2AF ( f , N.B1 + AF). Cell lysates prepared at the indicated times after G1/S release were subjected to immunoblotting with antibodies against cyclin B1 ( left ), or antibodies against the PSTAIRE sequence conserved among CDKs ( middle ; Cdc2 is the major anti-PSTAIRE antigen in HeLa cells). Arrowheads indicate the epitope-tagged, virally-encoded cyclin B1, NLS-cyclin B1, and Cdc2AF proteins. Histone H1 kinase activity was measured in immunoprecipitates with anti-cyclin B1 ( right ); autoradiographic exposure times were 1.5 h. ( C ) Cells from the same experiment as in B were harvested at the indicated times, fixed, stained with propidium iodide, and analyzed by flow cytometry to measure DNA content. Abbreviations are as given in B . ( D ) A fraction of the cells prepared for flow cytometric analysis in C were examined by microscopy for the presence of condensed chromosomes. At least 300 cells were analyzed for each sample. These values represent the means of data obtained from two separate experiments, in which results were essentially identical. Abbreviations are as given in B .

Techniques Used: Infection, Recombinant, Immunofluorescence, Sequencing, Activity Assay, Staining, Flow Cytometry, Cytometry, Microscopy

37) Product Images from "The Muscle Regulatory Factors MyoD and Myf-5 Undergo Distinct Cell Cycle-specific Expression in Muscle Cells "

Article Title: The Muscle Regulatory Factors MyoD and Myf-5 Undergo Distinct Cell Cycle-specific Expression in Muscle Cells

Journal: The Journal of Cell Biology

doi:

C2 myoblasts can be efficiently and accurately synchronized in a precise phase of their cell division cycle. Asynchronous C2 cells were arrested in quiescence by incubation in methionine depleted medium containing 1% serum for 36 h. Reentry into the cell cycle was allowed by refeeding them with proliferation medium. At different times after refeeding, cells were pulse-labeled for 15 min with BrdU before fixation and analysis for BrdU incorporation by immunofluorescence. ( A ) Plotted values represent the percentage of cells positive for BrdU incorporation ( empty circles ) and the percentage of mitotic cells ( filled squares ) over the total population of cells at given times after restimulation. ( B ) Cells, made quiescent by methionine deprivation, were induced to proliferate with serum containing medium and HU was added from 1 to 15 h after refeeding, allowing cells to progress through G1. Cells were subsequently released from HU block by washing off the HU and addition of serum containing medium, pulse-labeled for 15 min with BrdU and processed for immunofluorescence analysis at the indicated times. The percentage of cells in S phase was determined by counting BrdU positive cells over the total number of cells ( empty circles ). The percentage of mitotic cells is also plotted ( filled squares ). Percentages in A and B were determined after counting > 300 cells for each point. ( C ) cdk2 protein was immunoprecipitated from quiescent myoblasts at different times after release from methionine deprivation and from G1/S blocked myoblasts which were released 15 h after the addition of HU as in B (see Material and Methods). cdk2 kinase activity was evaluated against histone H1. Shown is an autoradiograph of the radioactivity incorporated into histone H1 and a Western blot analysis of the same membrane probed for anti-cdk2. Times above are in hours after refeeding. Synchronized cells with HU block are marked G1/S.
Figure Legend Snippet: C2 myoblasts can be efficiently and accurately synchronized in a precise phase of their cell division cycle. Asynchronous C2 cells were arrested in quiescence by incubation in methionine depleted medium containing 1% serum for 36 h. Reentry into the cell cycle was allowed by refeeding them with proliferation medium. At different times after refeeding, cells were pulse-labeled for 15 min with BrdU before fixation and analysis for BrdU incorporation by immunofluorescence. ( A ) Plotted values represent the percentage of cells positive for BrdU incorporation ( empty circles ) and the percentage of mitotic cells ( filled squares ) over the total population of cells at given times after restimulation. ( B ) Cells, made quiescent by methionine deprivation, were induced to proliferate with serum containing medium and HU was added from 1 to 15 h after refeeding, allowing cells to progress through G1. Cells were subsequently released from HU block by washing off the HU and addition of serum containing medium, pulse-labeled for 15 min with BrdU and processed for immunofluorescence analysis at the indicated times. The percentage of cells in S phase was determined by counting BrdU positive cells over the total number of cells ( empty circles ). The percentage of mitotic cells is also plotted ( filled squares ). Percentages in A and B were determined after counting > 300 cells for each point. ( C ) cdk2 protein was immunoprecipitated from quiescent myoblasts at different times after release from methionine deprivation and from G1/S blocked myoblasts which were released 15 h after the addition of HU as in B (see Material and Methods). cdk2 kinase activity was evaluated against histone H1. Shown is an autoradiograph of the radioactivity incorporated into histone H1 and a Western blot analysis of the same membrane probed for anti-cdk2. Times above are in hours after refeeding. Synchronized cells with HU block are marked G1/S.

Techniques Used: Incubation, Labeling, BrdU Incorporation Assay, Immunofluorescence, Blocking Assay, Immunoprecipitation, Activity Assay, Autoradiography, Radioactivity, Western Blot

38) Product Images from "Role of the casein kinase I isoform, Hrr25, and the cell cycle-regulatory transcription factor, SBF, in the transcriptional response to DNA damage in Saccharomyces cerevisiae"

Article Title: Role of the casein kinase I isoform, Hrr25, and the cell cycle-regulatory transcription factor, SBF, in the transcriptional response to DNA damage in Saccharomyces cerevisiae

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi:

In vitro  phosphorylation of Swi6 by Hrr25. ( A ) Kinase assays were done on eluates from Swi6 affinity columns or from control columns (no coupled protein). The presence of Swi6 on the column resin is indicated by a “+” above the lane, whereas “−” denotes the control column with no coupled protein. The columns were loaded with extracts from either a wild-type (lanes 1–7) or  hrr25 Δ strain (lanes 8 and 9) as indicated above the figure (“extract applied on column”). Exogenous substrate (100 ng) was added to the kinase assays as indicated above the lanes. mbp, myelin basic protein; H1, histone H1. ( B ) Kinase assays were done with 12CA5 (anti-HA) immunoprecipitates from yeast cells expressing an HA–Hrr25 fusion protein (lanes 1 and 3) or cells transformed with an empty vector (lane 2). In lanes 2 and 3, 100 ng of casein and Swi6 were added to the kinase reaction as indicated by a “+.” The position of migration of phosphorylated Swi6, Hrr25, and casein are indicated on the right. Molecular weight markers are shown on the left.
Figure Legend Snippet: In vitro phosphorylation of Swi6 by Hrr25. ( A ) Kinase assays were done on eluates from Swi6 affinity columns or from control columns (no coupled protein). The presence of Swi6 on the column resin is indicated by a “+” above the lane, whereas “−” denotes the control column with no coupled protein. The columns were loaded with extracts from either a wild-type (lanes 1–7) or hrr25 Δ strain (lanes 8 and 9) as indicated above the figure (“extract applied on column”). Exogenous substrate (100 ng) was added to the kinase assays as indicated above the lanes. mbp, myelin basic protein; H1, histone H1. ( B ) Kinase assays were done with 12CA5 (anti-HA) immunoprecipitates from yeast cells expressing an HA–Hrr25 fusion protein (lanes 1 and 3) or cells transformed with an empty vector (lane 2). In lanes 2 and 3, 100 ng of casein and Swi6 were added to the kinase reaction as indicated by a “+.” The position of migration of phosphorylated Swi6, Hrr25, and casein are indicated on the right. Molecular weight markers are shown on the left.

Techniques Used: In Vitro, Expressing, Transformation Assay, Plasmid Preparation, Migration, Molecular Weight

39) Product Images from "Coupled Integration of Human Immunodeficiency Virus Type 1 cDNA Ends by Purified Integrase In Vitro: Stimulation by the Viral Nucleocapsid Protein"

Article Title: Coupled Integration of Human Immunodeficiency Virus Type 1 cDNA Ends by Purified Integrase In Vitro: Stimulation by the Viral Nucleocapsid Protein

Journal: Journal of Virology

doi:

Stimulation of coupled joining by various nucleic acid binding proteins. (A) Products generated in the presence of 35 nM purified integrase and the indicated viral proteins. Expected structures of integration products are shown beside the gel. Maximum concentrations for each (right-most lane in each titration) are as follows: NC, 8 μg/ml (1 μM); MA, 16 μg/ml (0.94 μM); Rev, 4 μg/ml (0.2 μM); Tat, 4 μg/ml (0.28 μM); RT, 4 μg/ml (78 nM). Each protein was diluted 1:10 and 1:100 in the two left lanes. (B) Products generated in the presence of 35 nM purified integrase and the indicated cellular DNA binding proteins. Maximum concentrations for each (right-most lane in each titration) are as follows: NC, 8 μg/ml (1 μM); HMG I(Y), 16 μg/ml (1.4 μM); HMG-1, 4 μg/ml (0.16 μM); HMG-2, 8 μg/ml (0.64 μM); histone H1, 8 μg/ml (0.4 μM); Hu, 2.4 μg/ml (0.13 μM); BAF, 2 μg/ml (0.2 μM); RNase A, 4 μg/ml (0.3 μM); polylysine, 4 μg/ml (4 μM). Each protein was diluted 1:10 and 1:100 in the two left lanes.
Figure Legend Snippet: Stimulation of coupled joining by various nucleic acid binding proteins. (A) Products generated in the presence of 35 nM purified integrase and the indicated viral proteins. Expected structures of integration products are shown beside the gel. Maximum concentrations for each (right-most lane in each titration) are as follows: NC, 8 μg/ml (1 μM); MA, 16 μg/ml (0.94 μM); Rev, 4 μg/ml (0.2 μM); Tat, 4 μg/ml (0.28 μM); RT, 4 μg/ml (78 nM). Each protein was diluted 1:10 and 1:100 in the two left lanes. (B) Products generated in the presence of 35 nM purified integrase and the indicated cellular DNA binding proteins. Maximum concentrations for each (right-most lane in each titration) are as follows: NC, 8 μg/ml (1 μM); HMG I(Y), 16 μg/ml (1.4 μM); HMG-1, 4 μg/ml (0.16 μM); HMG-2, 8 μg/ml (0.64 μM); histone H1, 8 μg/ml (0.4 μM); Hu, 2.4 μg/ml (0.13 μM); BAF, 2 μg/ml (0.2 μM); RNase A, 4 μg/ml (0.3 μM); polylysine, 4 μg/ml (4 μM). Each protein was diluted 1:10 and 1:100 in the two left lanes.

Techniques Used: Binding Assay, Generated, Purification, Titration, DNA Binding Assay

40) Product Images from "Cell cycle arrest mediated by the MEK/mitogen-activated protein kinase pathway"

Article Title: Cell cycle arrest mediated by the MEK/mitogen-activated protein kinase pathway

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi:

), using histone H1 ( A ) or glutathione S -transferase-Rb (aa 379–928) ( B ) as substrates. Similar results were obtained in at least four independent experiments.
Figure Legend Snippet: ), using histone H1 ( A ) or glutathione S -transferase-Rb (aa 379–928) ( B ) as substrates. Similar results were obtained in at least four independent experiments.

Techniques Used:

Related Articles

Immunoprecipitation:

Article Title: Posttranslational Processing of Infected Cell Proteins 0 and 4 of Herpes Simplex Virus 1 Is Sequential and Reflects the Subcellular Compartment in Which the Proteins Localize
Article Snippet: .. Forty microliters of complete kinase buffer (50 mM Tris [pH 7.4], 10 mM MgCl2 , 5 mM DTT, 10 μM ATP, 20 μCi of [γ-32 P]ATP, 2 μg of histone H1) was added to immunoprecipitated cdk2 or cdc2. .. The samples were reacted at 30°C for 20 min, and the reaction was terminated by the addition of SDS-gel loading buffer (2% SDS, 5% β-mercaptoethanol, 50 mM Tris [pH 6.8], 2.75% sucrose) and heated to 95°C for 5 min.

Incubation:

Article Title: p21Waf1/Cip1 Inhibition of Cyclin E/Cdk2 Activity Prevents Endoreduplication after Mitotic Spindle Disruption
Article Snippet: .. The immunoprecipitates were washed twice with KLB and twice with kinase buffer (100 mM Tris [pH 7.4], 20 mM MgCl2 , 2 mM dithiothreitol) before being incubated with 5 μg of glutathione S -transferase (GST)-pRb (amino acids 792 to 928) ( ) (Cdk2, Cdk4, Cdk6, cyclin E) or 5 μg of histone H1 (Boehringer Mannheim) (cyclin B1) and 15 nM ATP for 10 min at 25°C. .. Samples were incubated with 2 μCi of [γ-32 P]ATP at 30°C for 10 min (Cdk2 and cyclin B1) or 10 μCi of [γ-32 P]ATP at 30°C for 30 min (Cdk4, Cdk6, and cyclin E), the reaction was stopped by addition of 2× Laemmli sample buffer, and the products were resolved by SDS-PAGE.

Article Title: The human papillomavirus E7 oncoprotein can uncouple cellular differentiation and proliferation in human keratinocytes by abrogating p21Cip1-mediated inhibition of cdk2
Article Snippet: .. The mixtures were incubated at 0°C for 10 min. One microgram of histone H1 (Boehringer Mannheim) and 10 μCi of [γ-32 P]ATP were added to initiate the kinase assay. ..

other:

Article Title: Induction of G2M Arrest by Flavokawain A, a Kava Chalcone, Increases the Responsiveness of HER2-Overexpressing Breast Cancer Cells to Herceptin
Article Snippet: Phosphorylation of histone H1 was measured by incubating the beads with 40 μL of “hot” kinase solution [0.25 μL (2.5 μg) of histone H1, 0.5 μL of [γ-32 P] ATP, 0.5 μL of 0.1 mM ATP, and 38.75 μL of kinase buffer] for 30 min at 37 °C.

Article Title: Identification of a substrate-targeting domain in cyclin E necessary for phosphorylation of the retinoblastoma protein
Article Snippet: Six of the 19 cyclin E mutants were markedly impaired in their ability to promote cell cycle progression, four of which retained the ability to activate CDK2 when assayed using histone H1 as a substrate (Table ).

Article Title: Direct Activation of Cyclin-Dependent Kinase 2 by Human Papillomavirus E7
Article Snippet: No cross-linking to histone H1 was detected for either peptide (Fig. ).

Activity Assay:

Article Title: Role of the casein kinase I isoform, Hrr25, and the cell cycle-regulatory transcription factor, SBF, in the transcriptional response to DNA damage in Saccharomyces cerevisiae
Article Snippet: .. The kinase activity seen in the Swi6 column eluates phosphorylated casein but not myelin basic protein or histone H1 (Fig. A , lanes 5–7). ..

Kinase Assay:

Article Title: The human papillomavirus E7 oncoprotein can uncouple cellular differentiation and proliferation in human keratinocytes by abrogating p21Cip1-mediated inhibition of cdk2
Article Snippet: .. The mixtures were incubated at 0°C for 10 min. One microgram of histone H1 (Boehringer Mannheim) and 10 μCi of [γ-32 P]ATP were added to initiate the kinase assay. ..

Article Title: Transgene Expression of bcl-xL Permits Anti-immunoglobulin (Ig)-induced Proliferation in xid B Cells
Article Snippet: .. The substrate for the kinase assay, histone H1, was purchased from Boehringer Mannheim (Indianapolis, IN). ..

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  • 92
    Boehringer Mannheim histone h1
    FKA increases MPM-2 phosphorylation and Cdc kinase activity via inhibition of Cdc2 and Cdc25 phosphorylation and downregulation of Myt1 and Wee1 expression. ( A ) Cdc2 associated <t>histone</t> H1 kinase activity was decreased dose-dependently by FKA treatment for 24 h in SKBR3 cells; ( B – D ) SKBR3 cells were treated with indicated concentrations of FKA for 24 h. Phosphorylation levels of Cdc2, MPM-2, and Cdc25C as well as protein levels of cyclin B1, Myt1, and Wee1 were detected by Western blotting analysis. β-actin was detected as a loading control. A representative blot was shown from three independent experiments.
    Histone H1, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 92/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Boehringer Mannheim hela nuclear extract
    Role of TBL1 in repression. ( a ) TBL1 potentiates repression by thyroid hormone receptor. A Gal4–TK-luciferase reporter was cotransfected with either Gal4 DBD, Gal4–TR, or Gal4–TR(V230R) and increasing amounts (black ramps representing 0.25–1.5 μg of DNA) of pCMX–HA–TBL1 into <t>HeLa</t> cells. Fold repression was measured as relative to Gal4 DBD alone and the results of duplicate samples are shown. ( b ) SMRT recruits TBL1 to TR. <t>GST-pulldown</t> assay using GST fusion to TRβ ligand-binding domain with 35 S-labeled TBL1 in the presence of control RRL (lanes 2,3 ) or unlabeled SMRT translated in RRL (lanes 4,5 ). ( c ) TBL1 contains an autonomous repression domain. Gal4–TBL1(1–577), Gal4–TBL1(1–211), and Gal4–TBL1(211–577) were transfected with the Gal4–TK-luciferase reporter and fold repression was measured relative to Gal4 DBD alone. ( d ) GST–TBL1 interacts with histone H3. HeLa Nuclear extract was incubated with either GST alone or GST–TBL1 and interacting histone H3 visualized by immunoblot.
    Hela Nuclear Extract, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hela nuclear extract/product/Boehringer Mannheim
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    FKA increases MPM-2 phosphorylation and Cdc kinase activity via inhibition of Cdc2 and Cdc25 phosphorylation and downregulation of Myt1 and Wee1 expression. ( A ) Cdc2 associated histone H1 kinase activity was decreased dose-dependently by FKA treatment for 24 h in SKBR3 cells; ( B – D ) SKBR3 cells were treated with indicated concentrations of FKA for 24 h. Phosphorylation levels of Cdc2, MPM-2, and Cdc25C as well as protein levels of cyclin B1, Myt1, and Wee1 were detected by Western blotting analysis. β-actin was detected as a loading control. A representative blot was shown from three independent experiments.

    Journal: Molecules (Basel, Switzerland)

    Article Title: Induction of G2M Arrest by Flavokawain A, a Kava Chalcone, Increases the Responsiveness of HER2-Overexpressing Breast Cancer Cells to Herceptin

    doi: 10.3390/molecules22030462

    Figure Lengend Snippet: FKA increases MPM-2 phosphorylation and Cdc kinase activity via inhibition of Cdc2 and Cdc25 phosphorylation and downregulation of Myt1 and Wee1 expression. ( A ) Cdc2 associated histone H1 kinase activity was decreased dose-dependently by FKA treatment for 24 h in SKBR3 cells; ( B – D ) SKBR3 cells were treated with indicated concentrations of FKA for 24 h. Phosphorylation levels of Cdc2, MPM-2, and Cdc25C as well as protein levels of cyclin B1, Myt1, and Wee1 were detected by Western blotting analysis. β-actin was detected as a loading control. A representative blot was shown from three independent experiments.

    Article Snippet: Phosphorylation of histone H1 was measured by incubating the beads with 40 μL of “hot” kinase solution [0.25 μL (2.5 μg) of histone H1, 0.5 μL of [γ-32 P] ATP, 0.5 μL of 0.1 mM ATP, and 38.75 μL of kinase buffer] for 30 min at 37 °C.

    Techniques: Activity Assay, Inhibition, Expressing, Western Blot

    Cross-linking of E7 peptides to CDK2/cyclin A complex. (A) HPV16 E7 (1.8 μM) and E7-derived peptides (50 μM) stimulate CDK2/cyclin A histone kinase activity. (B) Benzo-CYS-9-48-bio and benzo-NH 2 -9-48-bio peptides associate with cyclin A/CDK2 complex following UV irradiation. Histone H1 is not cross-linked under identical conditions. (C) CDK2/cyclin A (0.1 μM) and benzo-CYS-9-48-bio (0.5 μM) were with or without competitor E7 or E7 peptide. Both full-length 16E7 and the 9-48 16E7 peptide compete with the benzo-CYS-9-48-bio peptide for binding to the cyclin A/CDK2 complex. These results suggest that labeling of the CDK2/cyclin A complex by benzo-CYS-9-48-bio is specific and is not due to indiscriminate labeling of the complex. (D) Immunoprecipitation (IP) followed by Western blotting for incorporation of biotin-containing peptide into immunoprecipitated target protein(s) demonstrates that biotin-reactive bands in Western blots are cyclin A and CDK2 and that reactivity of these bands with benzo-CYS-9-48-bio peptide is dependent upon UV. (E) Cross-linking of benzophenone-containing peptides to increasing amounts of GST, cyclin A, or CDK2 demonstrates a strong preference of the peptide for cyclin A.

    Journal: Journal of Virology

    Article Title: Direct Activation of Cyclin-Dependent Kinase 2 by Human Papillomavirus E7

    doi: 10.1128/JVI.77.19.10566-10574.2003

    Figure Lengend Snippet: Cross-linking of E7 peptides to CDK2/cyclin A complex. (A) HPV16 E7 (1.8 μM) and E7-derived peptides (50 μM) stimulate CDK2/cyclin A histone kinase activity. (B) Benzo-CYS-9-48-bio and benzo-NH 2 -9-48-bio peptides associate with cyclin A/CDK2 complex following UV irradiation. Histone H1 is not cross-linked under identical conditions. (C) CDK2/cyclin A (0.1 μM) and benzo-CYS-9-48-bio (0.5 μM) were with or without competitor E7 or E7 peptide. Both full-length 16E7 and the 9-48 16E7 peptide compete with the benzo-CYS-9-48-bio peptide for binding to the cyclin A/CDK2 complex. These results suggest that labeling of the CDK2/cyclin A complex by benzo-CYS-9-48-bio is specific and is not due to indiscriminate labeling of the complex. (D) Immunoprecipitation (IP) followed by Western blotting for incorporation of biotin-containing peptide into immunoprecipitated target protein(s) demonstrates that biotin-reactive bands in Western blots are cyclin A and CDK2 and that reactivity of these bands with benzo-CYS-9-48-bio peptide is dependent upon UV. (E) Cross-linking of benzophenone-containing peptides to increasing amounts of GST, cyclin A, or CDK2 demonstrates a strong preference of the peptide for cyclin A.

    Article Snippet: No cross-linking to histone H1 was detected for either peptide (Fig. ).

    Techniques: Derivative Assay, Activity Assay, Irradiation, Binding Assay, Labeling, Immunoprecipitation, Western Blot

    Addition of increasing amounts of GST-16E7 to CDK2 purified from insect cells or from bacteria results in a dose-dependent increase in kinase activity. Autoradiograph (A) demonstrates E7-dependent increase in phosphorylation of histone H1 by CDK2/cyclin A. Quantification of the experiment (B) plots PhosphorImager counts versus amount of GST-16E7 added. Results indicate that E7 affect peaks at 640 ng of E7 (or approximately 0.6 μM E7). CDK2/cyclin A reconstituted from bacterially expressed proteins is also activated by purified 16E7 (C).

    Journal: Journal of Virology

    Article Title: Direct Activation of Cyclin-Dependent Kinase 2 by Human Papillomavirus E7

    doi: 10.1128/JVI.77.19.10566-10574.2003

    Figure Lengend Snippet: Addition of increasing amounts of GST-16E7 to CDK2 purified from insect cells or from bacteria results in a dose-dependent increase in kinase activity. Autoradiograph (A) demonstrates E7-dependent increase in phosphorylation of histone H1 by CDK2/cyclin A. Quantification of the experiment (B) plots PhosphorImager counts versus amount of GST-16E7 added. Results indicate that E7 affect peaks at 640 ng of E7 (or approximately 0.6 μM E7). CDK2/cyclin A reconstituted from bacterially expressed proteins is also activated by purified 16E7 (C).

    Article Snippet: No cross-linking to histone H1 was detected for either peptide (Fig. ).

    Techniques: Purification, Activity Assay, Autoradiography

    GST-16E7 activates purified cyclin E/CDK2 and cyclin A/CDK2 complexes. Autoradiographs demonstrate phosphorylation of histone H1 and Rb (pRb) by CDK2/cyclin E (A) and CDK2/cyclin A (B). (A) E7 potently upregulates CDK2/cyclin E histone H1 kinase activity while having modest effects on Rb kinase activity. Addition of p27 eliminates this effect and reduces activity to below baseline levels. (B) CDK2/cyclin A histone kinase activity is also dramatically upregulated by E7, and the effect is negated by addition of p27.

    Journal: Journal of Virology

    Article Title: Direct Activation of Cyclin-Dependent Kinase 2 by Human Papillomavirus E7

    doi: 10.1128/JVI.77.19.10566-10574.2003

    Figure Lengend Snippet: GST-16E7 activates purified cyclin E/CDK2 and cyclin A/CDK2 complexes. Autoradiographs demonstrate phosphorylation of histone H1 and Rb (pRb) by CDK2/cyclin E (A) and CDK2/cyclin A (B). (A) E7 potently upregulates CDK2/cyclin E histone H1 kinase activity while having modest effects on Rb kinase activity. Addition of p27 eliminates this effect and reduces activity to below baseline levels. (B) CDK2/cyclin A histone kinase activity is also dramatically upregulated by E7, and the effect is negated by addition of p27.

    Article Snippet: No cross-linking to histone H1 was detected for either peptide (Fig. ).

    Techniques: Purification, Activity Assay

    p21 induction in p53-deficient cells during MTI-induced endoreduplication reduces cyclin B1/Cdc2 kinase activity. (A) Western analysis of cyclin B1 and Cdc2. Asynchronous cells were treated with nocodazole (83 nM) for the indicated times, and protein was harvested. (B) Cyclin B1-associated kinase activity in the presence and absence of muristerone (7.5 μM) in nocodazole-treated HIp21 cells. Anti-cyclin B1 antibody was used to immunoprecipitate kinase complexes; histone H1 was used as a substrate. Quantification of the autoradiogram signals is presented in the histograms. Results are representative of three independent experiments.

    Journal: Molecular and Cellular Biology

    Article Title: p21Waf1/Cip1 Inhibition of Cyclin E/Cdk2 Activity Prevents Endoreduplication after Mitotic Spindle Disruption

    doi:

    Figure Lengend Snippet: p21 induction in p53-deficient cells during MTI-induced endoreduplication reduces cyclin B1/Cdc2 kinase activity. (A) Western analysis of cyclin B1 and Cdc2. Asynchronous cells were treated with nocodazole (83 nM) for the indicated times, and protein was harvested. (B) Cyclin B1-associated kinase activity in the presence and absence of muristerone (7.5 μM) in nocodazole-treated HIp21 cells. Anti-cyclin B1 antibody was used to immunoprecipitate kinase complexes; histone H1 was used as a substrate. Quantification of the autoradiogram signals is presented in the histograms. Results are representative of three independent experiments.

    Article Snippet: The immunoprecipitates were washed twice with KLB and twice with kinase buffer (100 mM Tris [pH 7.4], 20 mM MgCl2 , 2 mM dithiothreitol) before being incubated with 5 μg of glutathione S -transferase (GST)-pRb (amino acids 792 to 928) ( ) (Cdk2, Cdk4, Cdk6, cyclin E) or 5 μg of histone H1 (Boehringer Mannheim) (cyclin B1) and 15 nM ATP for 10 min at 25°C.

    Techniques: Activity Assay, Western Blot

    Loss of p21 results in cyclical Cdc2 kinase activity during MTI-induced endoreduplication. (A) Western analysis of cyclin B1 and Cdc2 proteins. Asynchronous cells were treated with nocodazole (83 nM) for the indicated times, and protein was harvested. (B) Cyclin B1-associated kinase activity in HCT116 p21 +/+ and p21 −/− cells. Anti-cyclin B1 antibody was used to immunoprecipitate kinase complexes; histone H1 was used as a substrate. Quantification of the autoradiogram signals is presented in the histograms. Results are representative of three independent experiments. (C) Whole-cell lysates from HCT116 p21 +/+ cells were immunoprecipitated with anti-cyclin B1, separated by SDS-PAGE, and analyzed for coimmunoprecipitation of p21 by Western blot analysis. Con, control representing p21 protein coimmunoprecipitated with anti-cyclin B1 from a whole-cell extract of WI-38 fibroblasts.

    Journal: Molecular and Cellular Biology

    Article Title: p21Waf1/Cip1 Inhibition of Cyclin E/Cdk2 Activity Prevents Endoreduplication after Mitotic Spindle Disruption

    doi:

    Figure Lengend Snippet: Loss of p21 results in cyclical Cdc2 kinase activity during MTI-induced endoreduplication. (A) Western analysis of cyclin B1 and Cdc2 proteins. Asynchronous cells were treated with nocodazole (83 nM) for the indicated times, and protein was harvested. (B) Cyclin B1-associated kinase activity in HCT116 p21 +/+ and p21 −/− cells. Anti-cyclin B1 antibody was used to immunoprecipitate kinase complexes; histone H1 was used as a substrate. Quantification of the autoradiogram signals is presented in the histograms. Results are representative of three independent experiments. (C) Whole-cell lysates from HCT116 p21 +/+ cells were immunoprecipitated with anti-cyclin B1, separated by SDS-PAGE, and analyzed for coimmunoprecipitation of p21 by Western blot analysis. Con, control representing p21 protein coimmunoprecipitated with anti-cyclin B1 from a whole-cell extract of WI-38 fibroblasts.

    Article Snippet: The immunoprecipitates were washed twice with KLB and twice with kinase buffer (100 mM Tris [pH 7.4], 20 mM MgCl2 , 2 mM dithiothreitol) before being incubated with 5 μg of glutathione S -transferase (GST)-pRb (amino acids 792 to 928) ( ) (Cdk2, Cdk4, Cdk6, cyclin E) or 5 μg of histone H1 (Boehringer Mannheim) (cyclin B1) and 15 nM ATP for 10 min at 25°C.

    Techniques: Activity Assay, Western Blot, Immunoprecipitation, SDS Page

    Role of TBL1 in repression. ( a ) TBL1 potentiates repression by thyroid hormone receptor. A Gal4–TK-luciferase reporter was cotransfected with either Gal4 DBD, Gal4–TR, or Gal4–TR(V230R) and increasing amounts (black ramps representing 0.25–1.5 μg of DNA) of pCMX–HA–TBL1 into HeLa cells. Fold repression was measured as relative to Gal4 DBD alone and the results of duplicate samples are shown. ( b ) SMRT recruits TBL1 to TR. GST-pulldown assay using GST fusion to TRβ ligand-binding domain with 35 S-labeled TBL1 in the presence of control RRL (lanes 2,3 ) or unlabeled SMRT translated in RRL (lanes 4,5 ). ( c ) TBL1 contains an autonomous repression domain. Gal4–TBL1(1–577), Gal4–TBL1(1–211), and Gal4–TBL1(211–577) were transfected with the Gal4–TK-luciferase reporter and fold repression was measured relative to Gal4 DBD alone. ( d ) GST–TBL1 interacts with histone H3. HeLa Nuclear extract was incubated with either GST alone or GST–TBL1 and interacting histone H3 visualized by immunoblot.

    Journal: Genes & Development

    Article Title: A core SMRT corepressor complex containing HDAC3 and TBL1, a WD40-repeat protein linked to deafness

    doi:

    Figure Lengend Snippet: Role of TBL1 in repression. ( a ) TBL1 potentiates repression by thyroid hormone receptor. A Gal4–TK-luciferase reporter was cotransfected with either Gal4 DBD, Gal4–TR, or Gal4–TR(V230R) and increasing amounts (black ramps representing 0.25–1.5 μg of DNA) of pCMX–HA–TBL1 into HeLa cells. Fold repression was measured as relative to Gal4 DBD alone and the results of duplicate samples are shown. ( b ) SMRT recruits TBL1 to TR. GST-pulldown assay using GST fusion to TRβ ligand-binding domain with 35 S-labeled TBL1 in the presence of control RRL (lanes 2,3 ) or unlabeled SMRT translated in RRL (lanes 4,5 ). ( c ) TBL1 contains an autonomous repression domain. Gal4–TBL1(1–577), Gal4–TBL1(1–211), and Gal4–TBL1(211–577) were transfected with the Gal4–TK-luciferase reporter and fold repression was measured relative to Gal4 DBD alone. ( d ) GST–TBL1 interacts with histone H3. HeLa Nuclear extract was incubated with either GST alone or GST–TBL1 and interacting histone H3 visualized by immunoblot.

    Article Snippet: Purification of histones from nuclear extract was performed by incubating 500 μg of HeLa nuclear extract with 1 μg of GST fusion protein in PBS containing 10% glycerol and protease inhibitors (Boehringer Mannheim) at 4°C overnight.

    Techniques: Luciferase, GST Pulldown Assay, Ligand Binding Assay, Labeling, Transfection, Incubation