Journal: PLoS ONE

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

doi: 10.1371/journal.pone.0028602

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

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

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

Journal: PLoS ONE

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

doi: 10.1371/journal.pone.0028602

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

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

Techniques: Activity Assay

Journal: PLoS ONE

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

doi: 10.1371/journal.pone.0028602

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

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

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

Journal: PLoS ONE

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

doi: 10.1371/journal.pone.0028602

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

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

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

Journal: PLoS ONE

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

doi: 10.1371/journal.pone.0028602

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

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

Techniques: Activity Assay

Journal: PLoS ONE

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

doi: 10.1371/journal.pone.0028602

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

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

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

Journal: PLoS ONE

Article Title: c-Rel Deficiency Increases Caspase-4 Expression and Leads to ER Stress and Necrosis in EBV-Transformed Cells

doi: 10.1371/journal.pone.0025467

Figure Lengend Snippet: ( A ) Pt1- (dashed line) and control D11-LCL tet cells (solid line) grown in the absence of Tc for 5 days were plated at 1×10 6 cells/mL in Tc media and cultured in parallel for 6 days. Cells were withdrawn at days 1, 3 and 6 cells and counted using Trypan-Blue exclusion to determine the total number of viable cells. ( B ) 1×10 6 Pt1- and control D11- and C2-LCL tet cells were stained with CFSE and cultured in Tc media (pink line), absence of Tc (for 5 days prior to CFSE staining) (grey peak) or absence of Tc plus sCD40L (500 ng/mL) (green line) for 6 days. At day 4 (upper panel) and day 6 (lower panel) cells were analyzed for proliferation by flow cytometry. ( C ) Pt1- and control D11-LCL tet cells (2×10 5 ) grown in Tc media were analyzed for the expression of phosphorylated-CDC2 by intra-cellular staining. Numbers shown are the mean fluorescence intensity (MFI) of cells expressing high levels of phosphorylated CDC2. Graph is representative of three independent experiments.

Article Snippet: Anti-S6 mAb, rabbit polyclonal anti-caspase 4 and rabbit polyclonal anti-phospho-CDC2 were purchased from Cell Signaling.

Techniques: Cell Culture, Staining, Flow Cytometry, Expressing, Fluorescence

Journal: Retrovirology

Article Title: Human T-lymphotropic virus type-1 p30 alters cell cycle G2 regulation of T lymphocytes to enhance cell survival

doi: 10.1186/1742-4690-4-49

Figure Lengend Snippet: p30 enhances phosphorylation of Cdc25C at S-216 . A) 40 ug of nuclear (N) or cytosolic (C) fraction prepared from either p30 expressing or Mock Jurkat T-cells were loaded on 10% SDS gel and western blot analysis was performed with anti-HA to confirm p30 expression and Histone H1 western to confirm nuclear and cytosolic fractionation. B) Western blot of nuclear (N) and cytosolic (C) extracts prepared from p30 or mock Jurkat T-cells probed with anti-Cdc2 and phosphospecific Cdc2 antibody. C) Western blot of nuclear (N) and cytosolic (C) extracts prepared from p30 or mock Jurkat T-cells, probed with anti-Cdc25C or phosphospecific anti-Cdc25C. D) Western Blot analysis of nuclear or cytosolic extracts from p30 or mock Jurkat T-cells, probed with anti-Chk1 or monoclonal phosphospecific Chk1 (S-345).

Article Snippet: Immunodetection was performed using the following antibodies: mouse anti-HA monoclonal antibody clone 16B-12 (1:1000, Covance Research Products, Princeton, NJ), mouse monoclonal anti-PARP clone C-2-10 (1:1000, Oncogene Research Products, Boston, MA). mouse monoclonal anti-β-actin clone AC-74 (1:4000, Sigma), rabbit polyclonal anti-Cdc2 # 9112 (1:1000, Cell Signaling, Beverly, MA), rabbit polyclonal anti-PLK1pT210 # 600-401-466 (1: 1000, Rockland, Gilbertsville, PA), polyclonal rabbit anti-Cdc2 T-161 #9114 (1:1000, Cell Signaling), polyclonal rabbit anti Cdc2 Y-15 #9111(1:1000, Cell Signaling), polyclonal rabbit anti-Cdc25C #9522 (1:1000, Cell Signaling), polyclonal rabbit anti-Cdc25C S-216 # 9528(1:1000, Cell Signaling), rabbit polyclonal anti-Cdc25C pS-198 # 9529 (1: 1000, Cell Signaling), polyclonal rabbit anti-Chk1 #2345 (1:1000, Cell Signaling), polyclonal rabbit anti-Chk1 S-345 #2341 (1: 1000, Cell Signaling), rabbit polyclonal anti-PLK1 (1: 1000) (Abcam, Cambridge, MA), mouse monoclonal anti-Histone H1, clone AE-4 (1: 1000, Upstate).

Techniques: Expressing, SDS-Gel, Western Blot, Fractionation

Journal: Retrovirology

Article Title: Human T-lymphotropic virus type-1 p30 alters cell cycle G2 regulation of T lymphocytes to enhance cell survival

doi: 10.1186/1742-4690-4-49

Figure Lengend Snippet: p30 enhances phosphorylation of Cdc25C at S-216 . A) 40 ug of nuclear (N) or cytosolic (C) fraction prepared from either p30 expressing or Mock Jurkat T-cells were loaded on 10% SDS gel and western blot analysis was performed with anti-HA to confirm p30 expression and Histone H1 western to confirm nuclear and cytosolic fractionation. B) Western blot of nuclear (N) and cytosolic (C) extracts prepared from p30 or mock Jurkat T-cells probed with anti-Cdc2 and phosphospecific Cdc2 antibody. C) Western blot of nuclear (N) and cytosolic (C) extracts prepared from p30 or mock Jurkat T-cells, probed with anti-Cdc25C or phosphospecific anti-Cdc25C. D) Western Blot analysis of nuclear or cytosolic extracts from p30 or mock Jurkat T-cells, probed with anti-Chk1 or monoclonal phosphospecific Chk1 (S-345).

Article Snippet: Immunodetection was performed using the following antibodies: mouse anti-HA monoclonal antibody clone 16B-12 (1:1000, Covance Research Products, Princeton, NJ), mouse monoclonal anti-PARP clone C-2-10 (1:1000, Oncogene Research Products, Boston, MA). mouse monoclonal anti-β-actin clone AC-74 (1:4000, Sigma), rabbit polyclonal anti-Cdc2 # 9112 (1:1000, Cell Signaling, Beverly, MA), rabbit polyclonal anti-PLK1pT210 # 600-401-466 (1: 1000, Rockland, Gilbertsville, PA), polyclonal rabbit anti-Cdc2 T-161 #9114 (1:1000, Cell Signaling), polyclonal rabbit anti Cdc2 Y-15 #9111(1:1000, Cell Signaling), polyclonal rabbit anti-Cdc25C #9522 (1:1000, Cell Signaling), polyclonal rabbit anti-Cdc25C S-216 # 9528(1:1000, Cell Signaling), rabbit polyclonal anti-Cdc25C pS-198 # 9529 (1: 1000, Cell Signaling), polyclonal rabbit anti-Chk1 #2345 (1:1000, Cell Signaling), polyclonal rabbit anti-Chk1 S-345 #2341 (1: 1000, Cell Signaling), rabbit polyclonal anti-PLK1 (1: 1000) (Abcam, Cambridge, MA), mouse monoclonal anti-Histone H1, clone AE-4 (1: 1000, Upstate).

Techniques: Expressing, SDS-Gel, Western Blot, Fractionation

Journal: Retrovirology

Article Title: Human T-lymphotropic virus type-1 p30 alters cell cycle G2 regulation of T lymphocytes to enhance cell survival

doi: 10.1186/1742-4690-4-49

Figure Lengend Snippet: p30 enhances phosphorylation of Cdc25C at S-216 . A) 40 ug of nuclear (N) or cytosolic (C) fraction prepared from either p30 expressing or Mock Jurkat T-cells were loaded on 10% SDS gel and western blot analysis was performed with anti-HA to confirm p30 expression and Histone H1 western to confirm nuclear and cytosolic fractionation. B) Western blot of nuclear (N) and cytosolic (C) extracts prepared from p30 or mock Jurkat T-cells probed with anti-Cdc2 and phosphospecific Cdc2 antibody. C) Western blot of nuclear (N) and cytosolic (C) extracts prepared from p30 or mock Jurkat T-cells, probed with anti-Cdc25C or phosphospecific anti-Cdc25C. D) Western Blot analysis of nuclear or cytosolic extracts from p30 or mock Jurkat T-cells, probed with anti-Chk1 or monoclonal phosphospecific Chk1 (S-345).

Article Snippet: Immunodetection was performed using the following antibodies: mouse anti-HA monoclonal antibody clone 16B-12 (1:1000, Covance Research Products, Princeton, NJ), mouse monoclonal anti-PARP clone C-2-10 (1:1000, Oncogene Research Products, Boston, MA). mouse monoclonal anti-β-actin clone AC-74 (1:4000, Sigma), rabbit polyclonal anti-Cdc2 # 9112 (1:1000, Cell Signaling, Beverly, MA), rabbit polyclonal anti-PLK1pT210 # 600-401-466 (1: 1000, Rockland, Gilbertsville, PA), polyclonal rabbit anti-Cdc2 T-161 #9114 (1:1000, Cell Signaling), polyclonal rabbit anti Cdc2 Y-15 #9111(1:1000, Cell Signaling), polyclonal rabbit anti-Cdc25C #9522 (1:1000, Cell Signaling), polyclonal rabbit anti-Cdc25C S-216 # 9528(1:1000, Cell Signaling), rabbit polyclonal anti-Cdc25C pS-198 # 9529 (1: 1000, Cell Signaling), polyclonal rabbit anti-Chk1 #2345 (1:1000, Cell Signaling), polyclonal rabbit anti-Chk1 S-345 #2341 (1: 1000, Cell Signaling), rabbit polyclonal anti-PLK1 (1: 1000) (Abcam, Cambridge, MA), mouse monoclonal anti-Histone H1, clone AE-4 (1: 1000, Upstate).

Techniques: Expressing, SDS-Gel, Western Blot, Fractionation

Journal: Retrovirology

Article Title: Human T-lymphotropic virus type-1 p30 alters cell cycle G2 regulation of T lymphocytes to enhance cell survival

doi: 10.1186/1742-4690-4-49

Figure Lengend Snippet: p30 enhances phosphorylation of Cdc25C at S-216 . A) 40 ug of nuclear (N) or cytosolic (C) fraction prepared from either p30 expressing or Mock Jurkat T-cells were loaded on 10% SDS gel and western blot analysis was performed with anti-HA to confirm p30 expression and Histone H1 western to confirm nuclear and cytosolic fractionation. B) Western blot of nuclear (N) and cytosolic (C) extracts prepared from p30 or mock Jurkat T-cells probed with anti-Cdc2 and phosphospecific Cdc2 antibody. C) Western blot of nuclear (N) and cytosolic (C) extracts prepared from p30 or mock Jurkat T-cells, probed with anti-Cdc25C or phosphospecific anti-Cdc25C. D) Western Blot analysis of nuclear or cytosolic extracts from p30 or mock Jurkat T-cells, probed with anti-Chk1 or monoclonal phosphospecific Chk1 (S-345).

Article Snippet: Immunodetection was performed using the following antibodies: mouse anti-HA monoclonal antibody clone 16B-12 (1:1000, Covance Research Products, Princeton, NJ), mouse monoclonal anti-PARP clone C-2-10 (1:1000, Oncogene Research Products, Boston, MA). mouse monoclonal anti-β-actin clone AC-74 (1:4000, Sigma), rabbit polyclonal anti-Cdc2 # 9112 (1:1000, Cell Signaling, Beverly, MA), rabbit polyclonal anti-PLK1pT210 # 600-401-466 (1: 1000, Rockland, Gilbertsville, PA), polyclonal rabbit anti-Cdc2 T-161 #9114 (1:1000, Cell Signaling), polyclonal rabbit anti Cdc2 Y-15 #9111(1:1000, Cell Signaling), polyclonal rabbit anti-Cdc25C #9522 (1:1000, Cell Signaling), polyclonal rabbit anti-Cdc25C S-216 # 9528(1:1000, Cell Signaling), rabbit polyclonal anti-Cdc25C pS-198 # 9529 (1: 1000, Cell Signaling), polyclonal rabbit anti-Chk1 #2345 (1:1000, Cell Signaling), polyclonal rabbit anti-Chk1 S-345 #2341 (1: 1000, Cell Signaling), rabbit polyclonal anti-PLK1 (1: 1000) (Abcam, Cambridge, MA), mouse monoclonal anti-Histone H1, clone AE-4 (1: 1000, Upstate).

Techniques: Expressing, SDS-Gel, Western Blot, Fractionation

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Leaf Extracts of Calocedrus formosana (Florin) Induce G2/M Cell Cycle Arrest and Apoptosis in Human Bladder Cancer Cells

doi: 10.1155/2011/380923

Figure Lengend Snippet: Western blot analysis of cell cycle regulatory protein levels and apoptosis proteins in TCCSUP cells treated with Florin extracts. (a) Cdc25C, Cdc-2, pCdc-2, cyclin-B1, and Bcl-2 proteins were analyzed after cells were exposed to Florin extracts. Cells were treated with 25 μ g/mL of Florin extracts for 6, 12, 24, and 36 h. Proteins (50 μ g) from each sample was resolved on 12% SDS-PAGE and Western blot was performed. GAPDH was used as a control. The thick and thin bars indicate the locations of the fast (dephosphorylated) and slow (phosphorylated) migrating forms of cyclin B1, P-Cdc2, or Cdc2, respectively. (b) Expression of Bcl-2 and Bax proteins in TCCSUP cells was analyzed. Cells were treated with 6, 12, and 25 μ g/mL of Florin extracts for 24 h. Proteins (50 μ g) from each sample were resolved on 12% SDS-PAGE and Western blot was performed. GAPDH was used as a control. The two slow migrating forms of Bcl2 may be the hyperphosphorylated forms of Bcl2.

Article Snippet: Cell lysates with equal amounts of proteins, which were measured using a BCA Protein Reagent Kit (Pierce, Rockford, IL, USA), were analyzed by Western blot, using a rabbit polyclonal antibody to cdc2 phosphorylated at Tyr15 (p-Cdc2) (1 : 1000; R&D, Minneapolis, MN, USA), a rabbit polyclonal cdc2 antibody (1 : 1000; Cell Signaling Technology, St. Louis, MO, USA), a mouse monoclonal antibody to cyclin B1 (sc-254; 1 : 200) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), a mouse monoclonal antibody to poly (ADP-ribose) polymerase (PARP) (0.5 μ g/mL; from BD Biosciences, Franklin Lakes, NJ, USA), a mouse monoclonal antibody to Bcl-2, a rabbit polyclonal antibody to Bax (1 : 200; both from DAKO, Taipei, Taiwan), an anti- β -tubulin mouse monoclonal antibody (1 : 20000; Epitomics, Burlingame, California), or a mouse monoclonal antibody against human GAPDH (1 : 1000; Santa Cruz, CA, USA).

Techniques: Western Blot, SDS Page, Expressing