Journal:

Article Title: Requirement for p27 KIP1 in Retinoblastoma Protein-Mediated Senescence

doi: 10.1128/MCB.21.11.3616-3631.2001

Figure Lengend Snippet: E2F repression is not required for pRb upregulation of p27KIP1. SAOS-2 cells were transiently transfected with empty vector (pSVE), RB, HA-pRbΔ651, HA-pRbΔ657, or HA-pRbΔ663. (A) Cells were cotransfected with an expression vector for the CD20 cell surface protein. Forty-eight hours posttransfection, cells were harvested, fixed, incubated with FITC-conjugated anti-CD20 to identify transfected cells, and stained with PI to determine DNA content. Cell cycle analysis was then performed by FACS, with 10,000 CD20-positive events counted. Results represent the percent increase of cells in the G1 phase over vector-transfected cells and are the average of at least two independent experiments. (B) Immunoblot of transfected cells 48 h posttransfection. (C) Immunoblot and in vitro kinase assay of lysates immunoprecipitated with anti-cyclin E 48 h after transfection. (D) Immunofluorescence analysis of cyclin E, cdk2, and p27KIP1 in transfected cells. Cells transfected with vector, RB, or HA-pRbΔ663 were coimmunostained with cyclin E (green), cdk2 (blue), and p27KIP1 (red) antibodies 48 h posttransfection.

Article Snippet: Primary antibodies used for staining were monoclonal anti-p27 K25050 (1:100 dilution) (Transduction Laboratories), goat polyclonal anti-cyclin E C-19 (1:50 dilution) (Santa Cruz), rabbit polyclonal anti-cdk2 M2 (1:50 dilution) (Santa Cruz), and monoclonal anti-α-tubulin (1:1,000 dilution) (Sigma).

Techniques: Transfection, Plasmid Preparation, Expressing, Incubation, Staining, Cell Cycle Assay, Western Blot, In Vitro, Kinase Assay, Immunoprecipitation, Immunofluorescence

Journal:

Article Title: Requirement for p27 KIP1 in Retinoblastoma Protein-Mediated Senescence

doi: 10.1128/MCB.21.11.3616-3631.2001

Figure Lengend Snippet: pRb pocket mutants induce senescence and p27KIP1 accumulation. SAOS-2 cells were transfected with empty vector (pSVE), RB, HA-p107, HA-p130, HA-pRbΔ651, HA-pRbΔ657, or HA-pRbΔ663 and a puromycin resistance plasmid. (A) Cells were puromycin selected 48 h after transfection and at 5 days posttransfection were labeled with BrdU and stained with anti-BrdU, and BrdU-positive cells were counted (at least 250 cells). Results represent the decrease in BrdU-positive cells compared to vector-transfected cells and are the average of at least three experiments. (B) Ten days after transfection puromycin-selected cells were stained for SA–β-gal activity and flat cells (solid bars) and SA–β-gal-positive cells (hatched bars) were counted. Results are the number of flat or SA–β-gal-positive cells divided by the total number of cells counted (at least 100 cells) and represent the average of at least three experiments. (C) Immunoblot at 10 days of lysates from cells transfected with the indicated plasmids and puromycin selected. (D) Ten days posttransfection cyclin E immunoprecipitation and in vitro kinase assay of cells transfected with the indicated plasmids and puromycin selected. (E) Coimmunostaining of vector-, RB-, or HA-pRbΔ663-transfected cells with cyclin E (green), cdk2 (blue), and p27KIP1 (red) antibodies 10 days posttransfection. Yellow staining in RB-transfected cells indicates colocalization of cyclin E and p27KIP1.

Article Snippet: Primary antibodies used for staining were monoclonal anti-p27 K25050 (1:100 dilution) (Transduction Laboratories), goat polyclonal anti-cyclin E C-19 (1:50 dilution) (Santa Cruz), rabbit polyclonal anti-cdk2 M2 (1:50 dilution) (Santa Cruz), and monoclonal anti-α-tubulin (1:1,000 dilution) (Sigma).

Techniques: Transfection, Plasmid Preparation, Labeling, Staining, Activity Assay, Western Blot, Immunoprecipitation, In Vitro, Kinase Assay

Journal:

Article Title: Requirement for p27 KIP1 in Retinoblastoma Protein-Mediated Senescence

doi: 10.1128/MCB.21.11.3616-3631.2001

Figure Lengend Snippet: p27KIP1 induces senescence. SAOS-2 cells were transfected with empty vector (pSVE), RB, p16INK4a, p21CIP1, p27KIP1, and dominant-negative Cdk2 (dnCdk2 or dnK2) expression vectors. (A) Cell cycle inhibitors were cotransfected with empty vector (solid bars) or with RB (hatched bars) and with pBabe-puro, selected with puromycin 24 h after transfection, and then stained for SA–β-gal activity 10 days posttransfection. (B) p27KIP1 immunoblot at 10 days of lysates from cells transfected with the indicated plasmids. (C) Phenotype of pRb and p27KIP1 SA–β-gal-positive cells 10 days posttransfection. (D) PAI-1 immunoblot of cells transfected with the indicated plasmids 10 days after transfection. (E) Effect of pRb and p27KIP1 on microtubulin in senescent cells. Ten days posttransfection cells were coimmunostained with p27KIP1 (green) and α-tubulin (red) antibodies.

Article Snippet: Primary antibodies used for staining were monoclonal anti-p27 K25050 (1:100 dilution) (Transduction Laboratories), goat polyclonal anti-cyclin E C-19 (1:50 dilution) (Santa Cruz), rabbit polyclonal anti-cdk2 M2 (1:50 dilution) (Santa Cruz), and monoclonal anti-α-tubulin (1:1,000 dilution) (Sigma).

Techniques: Transfection, Plasmid Preparation, Dominant Negative Mutation, Expressing, Staining, Activity Assay, Western Blot

Journal:

Article Title: Requirement for p27 KIP1 in Retinoblastoma Protein-Mediated Senescence

doi: 10.1128/MCB.21.11.3616-3631.2001

Figure Lengend Snippet: Model for pRb-mediated senescence. pRb represses E2F-mediated transcription of S-phase genes, inducing an acute cell cycle arrest. In a non-E2F-dependent manner, pRb upregulates p27KIP1 expression, leading to an accumulation of p27KIP1 levels and a persistent inhibition of cyclin E-cdk2 kinase activity. The specific inhibition of cyclin E kinase activity by the CIP/KIP inhibitors triggers senescence, but it is a unique function of pRb to induce the morphology change associated with senescent cells that appears to strongly correlate with levels of p27KIP1 expression.

Article Snippet: Primary antibodies used for staining were monoclonal anti-p27 K25050 (1:100 dilution) (Transduction Laboratories), goat polyclonal anti-cyclin E C-19 (1:50 dilution) (Santa Cruz), rabbit polyclonal anti-cdk2 M2 (1:50 dilution) (Santa Cruz), and monoclonal anti-α-tubulin (1:1,000 dilution) (Sigma).

Techniques: Expressing, Inhibition, Activity Assay

Journal: Oncogene

Article Title: DNA damage induces Chk1-dependent threonine-160 phosphorylation and activation of Cdk2.

doi: 10.1038/onc.2009.340

Figure Lengend Snippet: Figure 2 Requirement of cyclin-dependent kinase (Cdk)1 or Cdk2 for centrosome amplification in DT40 cells. (a) Immuno- fluorescence micrograph showing radiation-induced centrosome amplification in Cdk-deficient DT40 cells. Cells of the indicated genotype were fixed and stained 12 h after 10 Gy g-irradiation for centrin (green), g-tubulin (red) and counterstained with 1R,2R- diaminocyclohexane(trans-diacetato) (dichloro) platinum(IV) (DAPI) (blue). Scale bar, 10 mm. Cells were fixed and stained for g-tubulin as previously described (Bourke et al., 2007), also using polyclonal anti-centrin (#628802, Biolegend, Uithoorn, the Netherlands). Microscopy and centrosome analyses were as previously described (Bourke et al., 2007). (b) Quantitation of cells with aberrant centrosome numbers in cells of the indicated genotype before or 12 h after 10 Gy g-irradiation, based on immunofluorescence microscopy analysis of g-tubulin. Cdk1/Cdk1AS and Cdk1/Cdk1AS/Cdk2/ cells pretreated for 10 min with the Cdk1AS inhibitor 4-amino-1-tert-butyl-3-(10-naphthylmethyl)pyrazolo[3,4-d]pyrimidine (1NM-PP1) are labelled as Cdk1OFF (Hochegger et al., 2007). (c) Upregulation of Cdk2 activity in Cdk1OFF cells and Cdk1 activity in Cdk2/ cells. Immunoprecipitation-kinase reactions were carried out on cells of the indicated genotypes as described for Figure 1a. Immunoprecipitation controls from cells of the indicated genotypes are also shown. (d) Quantitation of cells with aberrant centrosome numbers in cells of the indicated genotype before or after 24 h 2 mM hydroxyurea (HU) treatment. Fixation, staining and quantitation were as described for (a) and (b).

Article Snippet: Immunoprecipitation was carried out with antibodies to Cdk2 (M2; sc-163, Santa Cruz, Heidelberg, Germany) or Cdk1 (sc-54; Santa Cruz).

Techniques: Staining, Irradiation, Microscopy, Quantitation Assay, Activity Assay, Immunoprecipitation

Journal:

Article Title: A novel role for the cyclin-dependent kinase inhibitor p27 Kip1 in angiotensin II-stimulated vascular smooth muscle cell hypertrophy

doi:

Figure Lengend Snippet: Ang II induces cell-cycle protein upregulation but not activation of Cdk’s. Confluent, quiescent VSMCs (time 0) were either stimulated with Ang II (100 nM) or 10% serum for up to 48 hours. (a) Cell-cycle protein expression was determined by immunoblot of whole-cell lysates. Histone H1 kinase assay was performed after immunoprecipitation (IP) with either anti-Cdk2 or anti-Cdk1, and phosphorylated histone H1 was viewed on a 12% SDS-PAGE. All blots were independently repeated at least 3 times. (b) Quantification of histone H1 kinase assay, for the 24-hour time point (n = 4; *P < 0.002).

Article Snippet: The following primary antibodies were used in the study: mouse monoclonal anti-Cdk1, mouse monoclonal anti-PCNA (PC10), rabbit polyclonal anti-Cdk2 (M2), mouse monoclonal anti-p27 Kip1 (F8), mouse monoclonal anti-cyclin D1 (HD11), and rabbit polyclonal anti-Cdk4 (C-22) (Santa Cruz Biotechnology Inc., Santa Cruz, California, USA).

Techniques: Activation Assay, Expressing, Western Blot, Kinase Assay, Immunoprecipitation, SDS Page

Journal:

Article Title: A novel role for the cyclin-dependent kinase inhibitor p27 Kip1 in angiotensin II-stimulated vascular smooth muscle cell hypertrophy

doi:

Figure Lengend Snippet: Ang II fails to downregulate the Cdk inhibitor p27Kip1, which binds to and inhibits Cdk2. Confluent, quiescent VSMCs (time 0) were either stimulated with Ang II (100 nM) or 10% serum for up to 24 hours. Immunoblots (IB) for p27Kip1 were done of whole-cell lysates (a) or after immunoprecipitation (IP) with anti-Cdk2 (b).

Article Snippet: The following primary antibodies were used in the study: mouse monoclonal anti-Cdk1, mouse monoclonal anti-PCNA (PC10), rabbit polyclonal anti-Cdk2 (M2), mouse monoclonal anti-p27 Kip1 (F8), mouse monoclonal anti-cyclin D1 (HD11), and rabbit polyclonal anti-Cdk4 (C-22) (Santa Cruz Biotechnology Inc., Santa Cruz, California, USA).

Techniques: Western Blot, Immunoprecipitation

Journal: Oncogene

Article Title: Characterization of an E2F-p130 complex formed during growth arrest.

doi: 10.1038/sj.onc.1201224

Figure Lengend Snippet: Figure 2 Analysis of E2F complexes from CV-1 cells by EMSA. Cell extracts were studied by EMSA using a 32P-labeled E2F- speci®c oligonucleotide probe either in the presence or absence of various antisera and analysed by polyacrylamide gel electrophoresis, as in Figure 1. Serum-starved (lanes 1 ± 6), hydroxyurea- (lanes 7 ± 12) or nocodazole-treated cells (lanes 13 ± 18). Lanes 1,7,13: no antibody; lanes 2,8,14: plus anti-pRB 896 antiserum; lanes 3,9,15: anti-p107 SD9 antibody; lanes 4,10,16; anti-p130 C20 antiserum; lanes 5,11,17: anti-cyclin A C160 antibody; lanes 6,12,18: anti-Cdk2 M-2 antiserum. The positions of free E2F species, C7 and other E2F complexes, and a background band (*) have been presented at the right and left

Article Snippet: Puri®ed antisera against p107 (SD9 and C-18), p130 (C-20) and Cdk2 (M-2) were purchased from Santa Cruz Biotechnology, and anti-pRB serum G3-245 from Pharmingen.

Techniques: Labeling, Polyacrylamide Gel Electrophoresis

Journal: Oncogene

Article Title: Characterization of an E2F-p130 complex formed during growth arrest.

doi: 10.1038/sj.onc.1201224

Figure Lengend Snippet: Figure 6 Analysis of E2F complexes following addition of recombinant proteins. Figure 7(a): Eect of Cdk and cyclin A on E2F complexes. E2F complexes present in serum-starved and asynchronously growing CV-1 cells were studied by EMSA as in Figure 1, following addition of recombinant GST-cyclin A, GST-Cdk2, or both. Lanes are as described in the Figure. Figure 7(b): Eect of adenovirus E1A protein on E2F complexes. E2F complexes present in serum-starved and asynchronous CV-1 cells and in terminally dierentiated mouse C2C12 myotubes were studied by EMSA as in Figure 1, following addition of in vitro translated wild-type E1A-243R, dl37 ± 68 mutant E1A-243R, or dl37 ± 68 E1A protein and E1A-speci®c M58 antibody. Lanes are as described in the Figure. For both Figure 7a and b the positions of free E2F species, C7 and E2F complexes, and a background band (*) have been presented at the right and left as has that of C*, the E2F complexes which were `supershifted' by M58 serum

Article Snippet: Puri®ed antisera against p107 (SD9 and C-18), p130 (C-20) and Cdk2 (M-2) were purchased from Santa Cruz Biotechnology, and anti-pRB serum G3-245 from Pharmingen.

Techniques: Recombinant, In Vitro, Mutagenesis

Journal: PLoS ONE

Article Title: Toxicological Profile of Ultrapure 2,2′,3,4,4′,5,5′-Heptachlorbiphenyl (PCB 180) in Adult Rats

doi: 10.1371/journal.pone.0104639

Figure Lengend Snippet: Tumor suppressor protein p53 and the DNA damage signaling markers were dose-dependently increased only in females. Each column represents mean ± SD (n = 5) as percent of control after adjustment to the loading control (Cdk2). Data was obtained from at least three independent analyses.

Article Snippet: Primary antibodies for DNA-damage marker were phospho-p53 (Ser-15), phospho-Chk2 (Thr-68), phospho-Mdm2 (Ser-166), phospho-Akt (Ser-473) (Cell Signaling Technology, Stockholm, Sweden) phospho-Erk (Tyr-204) (E-4) and the loading control Cdk2 (M2) (sc-7383 resp. sc-163, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Control