Journal: Oncogene

Article Title: ERK1/2 and p38 cooperate to induce a p21CIP1-dependent G1 cell cycle arrest.

doi: 10.1038/sj.onc.1207467

Figure Lengend Snippet: Figure 4 DMEKK3:ER* fails to induce p21CIP1 expression and causes only a transient delay in cell cyle re-entry in Rat-1 cells. (a) Quiescent CM3 and RM3 cells were treated with 10% FBS in the presence of ethanol or 100 nM 4-HT for the indicated times and whole-cell lysates were prepared, normalized for protein content and probed for p21CIP1 expression (b) (Left) Quiescent RM3.21 cells were treated with 10% FBS in the presence of ethanol or 100 nM 4- HT for the indicated times. CDK2 activity was measured by immune complex kinase assay and was expressed as radioactivity incorporated into histone H1 in phosphorimager (PI) units. Results are mean7range of n ¼ 2 experiments; similar results were obtained in a third experiment. Right: in addition, whole-cell lysates were analysed by immunoblot for phosphorylation of pRb and expression of p107 and cyclin A. Results are taken from a single experiment; similar results were observed in a total of three independent experiments. (c,d) Quiescent RM3.16 cells were stimulated with 10% FBS in the presence of ethanol or 100 nM 4- HT for increasing periods of time. (c) DNA synthesis was assayed by [3H]thymidine incorporation (mean7range of duplicate sam- ples) while in (d) cells were stained with propidium iodide and their DNA content was measured by flow cytometry. Results are taken from a single experiment; similar results were observed in a total of three independent experiments

Article Snippet: The following antibodies were used: Myc tag antibody (9E10); CDK2 (sc-163), CDK4 (sc260), Cdc25A (sc-7389), cyclin E (sc-481) and p107 (sc-318) were from Santa Cruz; cyclin D1 (CC12) and p27Kip1 (NA35) from Calbiochem; cyclin A (MS-384-P) and p53 (MS-104-P) from Neo Markers; p21Cip1 (556431) from Pharmingen; Rb Cterminal control antibody (9302), phospho-IkB (9241), IkB (9242), MAPKAP-K2 (3042), phospho-MAPKAP-K2 (3044) and phospho-ERK1/2 (9106) were from Cell Signalling Technology.

Techniques: Expressing, Activity Assay, Immune Complex Kinase Assay, Radioactivity, Western Blot, Phospho-proteomics, DNA Synthesis, Staining, Cytometry

Journal: Oncogene

Article Title: ERK1/2 and p38 cooperate to induce a p21CIP1-dependent G1 cell cycle arrest.

doi: 10.1038/sj.onc.1207467

Figure Lengend Snippet: Figure 5 p21CIP1 is required for DMEKK3:ER* to induce an optimal G1 arrest. (a) WT or p21CIP1/ 3T3 cells expressing Myc- DMEKK3:ER* were kept in complete medium (C) or serum starved overnight (SF) before restimulating with FBS in the presence of ethanol (F) or 100 nM 4-HT (F þ 4-HT) for 24 h. Whole-cell lysates were prepared, fractionated by SDS–PAGE and blotted with antibodies for the Myc tag on DMEKK3:ER*, phospho-c-Jun or p21CIP1. Similar results were seen in two independent experiments. (b) WT 3T3 cells were treated with 10% serum in the presence of ethanol or 100 nM 4-HT for the indicated times and whole-cell lysates were prepared, normalized for protein content and blotted for p21CIP1, cyclin A and p107. (c,d) WT and p21CIP1/ cells treated as above (a) were assayed for [3H]thymidine incorporation (c) or stained with propidium iodide and analysed for cell cycle distribution by flow cytometry (d). (c) Results are taken from a single experiment and represent the mean7s.d. of triplicate cell samples. The figures indicate the % inhibition of FBS-stimulated DNA synthesis by 4-HT in each cell line. (c) The percentage of cells in each phase of the cell cycle is indicated

Article Snippet: The following antibodies were used: Myc tag antibody (9E10); CDK2 (sc-163), CDK4 (sc260), Cdc25A (sc-7389), cyclin E (sc-481) and p107 (sc-318) were from Santa Cruz; cyclin D1 (CC12) and p27Kip1 (NA35) from Calbiochem; cyclin A (MS-384-P) and p53 (MS-104-P) from Neo Markers; p21Cip1 (556431) from Pharmingen; Rb Cterminal control antibody (9302), phospho-IkB (9241), IkB (9242), MAPKAP-K2 (3042), phospho-MAPKAP-K2 (3044) and phospho-ERK1/2 (9106) were from Cell Signalling Technology.

Techniques: Expressing, SDS Page, Staining, Cytometry, Inhibition, DNA Synthesis

Journal: Oncogene

Article Title: Rb and p130 regulate RNA polymerase I transcription: Rb disrupts the interaction between UBF and SL-1.

doi: 10.1038/sj.onc.1203875

Figure Lengend Snippet: Figure 6 UBF coimmunoprecipitates with p130 but not p107. A nuclear extract of N1S1 cells was incubated with either anti-p130 or anti-p107 antibodies and protein-A Sepharose. The immuno- precipitates were collected, washed, boiled in 26Laemmli sample buer, fractionated by SDS ± PAGE, and transferred to Immobi- lon-P. After UBF was detected with anti-UBF antiserum and ECL the ®lter was stripped, and p130 and p107 were detected with anti-p130 and p107 antibodies, respectively. The `load' (lane 1) represents 5% of the input protein and the supernatants (Super., lanes 4 and 5) contained approximately 10% of the unbound material before any washes

Article Snippet: Polyclonal anti-p130 (Cat# sc-317), monoclonal anti-TBP (Cat# sc-421) and monoclonal p107 (Cat# sc-318) were obtained from Santa Cruz (California).

Techniques: Incubation, SDS Page

Journal: Journal of Biological Chemistry

Article Title: A B-myb Promoter Corepressor Site Facilitatesin Vivo Occupation of the Adjacent E2F Site by p107·E2F and p130·E2F Complexes

doi: 10.1074/jbc.m202960200

Figure Lengend Snippet: FIG. 3. Transcriptional repression of the B-myb promoter by p107E2F complexes requires the DRS. A, the wild-type (wt) B-myb promoter/reporter pGL2-(536) or derivatives containing mutations in either the E2F (mE2F) or DRS (mDRS) site were transfected into primary MEFs derived from control p107/ or knockout p107// p130/ animals. Luciferase was assayed in transfected cells deprived of serum for 60 h. B, knockout p107//p130/ MEFs were transfected with the B-myb promoter/reporters described for A together with either the empty pCMV expression vector (control (Con)) or vector encoding p107 or Myc epitope-tagged p130. Luciferase was assayed in transfected cells deprived of serum for 60 h. Data are presented as -fold repression, calculated by dividing the average luciferase value obtained with the control empty vector by the average value obtained for that reporter with the p107 or p130 expression vector. The control for each reporter was assigned a value of unity.

Article Snippet: These were used in EMSAs as described above; however, the antibodies used in supershifts were the same as those used in ChIP assays (rabbit anti-p107 polyclonal antibody, Santa Cruz Biotechnology sc-318; rabbit anti-p130 polyclonal antibody, Santa Cruz Biotechnology sc-317; and anti-Rb monoclonal antibody 21C9).

Techniques: Transfection, Derivative Assay, Control, Knock-Out, Luciferase, Expressing, Plasmid Preparation

Journal: Journal of Biological Chemistry

Article Title: A B-myb Promoter Corepressor Site Facilitatesin Vivo Occupation of the Adjacent E2F Site by p107·E2F and p130·E2F Complexes

doi: 10.1074/jbc.m202960200

Figure Lengend Snippet: FIG. 4. The DRS is required for occupation of the B-myb E2F site with p130 and p107 complexes. A, stable NIH 3T3 transfectants were established with luciferase reporter genes driven by either the wild-type (wt) B-myb promoter or mutant B-myb promoters with nonfunctional E2F (mE2F) or DRS (mDRS) sites. The reporter genes were in each case flanked by scaffold/matrix attachment regions from the human interferon- gene to attenuate positional effects, and clones were selected with approximately five transgene copies. Luciferase activities were assayed in cells arrested in G0 by serum deprivation for 60 h and after restimulation with serum for 16 h (S phase). B, EMSA was performed with whole cell extracts prepared from NIH 3T3 cell clones stably transfected with the wild-type and mDRS B-myb promoter/luciferase reporters. Pocket proteinE2F complexes were supershifted with antibodies (Ab) against p107, p130, or Rb as indicated. Control reactions (Con) contained preimmune rabbit serum. C, NIH 3T3 cell clones stably transfected with the wild-type and mDRS B-myb promoter/luciferase reporters were arrested in G0 by serum deprivation, and then proteinDNA complexes were formaldehyde-cross-linked in vivo. Chromatin fragments from these cells were subjected to immunoprecipitation (IP) with control serum or with antibodies to p107, p130, or Rb as indicated. After cross-link reversal, the co-immunopre- cipitated DNA was amplified by PCR and detected by Southern blotting. Separate primers were used to detect the transgene and the endogenous gene. D, shown is the quantitation by phosphorimaging of the results shown in C for the wild-type promoter-transfected cell transgene (wt/Tr) and endogenous gene (wt/En) and the mDRS transgene (mDRS/Tr) and endogenous gene (mDRS/En).

Article Snippet: These were used in EMSAs as described above; however, the antibodies used in supershifts were the same as those used in ChIP assays (rabbit anti-p107 polyclonal antibody, Santa Cruz Biotechnology sc-318; rabbit anti-p130 polyclonal antibody, Santa Cruz Biotechnology sc-317; and anti-Rb monoclonal antibody 21C9).

Techniques: Luciferase, Mutagenesis, Clone Assay, Stable Transfection, Transfection, Control, In Vivo, Immunoprecipitation, Amplification, Southern Blot, Quantitation Assay

Journal: Journal of Biological Chemistry

Article Title: A B-myb Promoter Corepressor Site Facilitatesin Vivo Occupation of the Adjacent E2F Site by p107·E2F and p130·E2F Complexes

doi: 10.1074/jbc.m202960200

Figure Lengend Snippet: FIG. 5. Knockout p107//p130/ embryos contain abundant nuclear RbE2F complexes. Cytoplasmic (CYT) and nuclear (NUC) extracts were prepared from knockout p107//p130/ and control p107/ MEFs that were arrested in G0 by serum deprivation for 60 h. EMSA was performed with these extracts using a 32P-labeled oligonucleotide probe containing the B-myb E2F/DRS sites. Control (Con; preimmune rabbit serum), anti-p130, anti-Rb, anti-p107, or anti-E2F-4 antibodies were added to EMSA reactions as indicated. Arrowheads indicate the positions of various E2F complexes and antibody (Ab) supershifts. A, p107//p130/ MEF cytoplasmic extracts; B, p107//p130/ MEF nuclear extracts; C, p107/ MEF cytoplasmic extracts; D, p107/ MEF nuclear extracts.

Article Snippet: These were used in EMSAs as described above; however, the antibodies used in supershifts were the same as those used in ChIP assays (rabbit anti-p107 polyclonal antibody, Santa Cruz Biotechnology sc-318; rabbit anti-p130 polyclonal antibody, Santa Cruz Biotechnology sc-317; and anti-Rb monoclonal antibody 21C9).

Techniques: Knock-Out, Control, Labeling

Journal: Journal of Biological Chemistry

Article Title: A B-myb Promoter Corepressor Site Facilitatesin Vivo Occupation of the Adjacent E2F Site by p107·E2F and p130·E2F Complexes

doi: 10.1074/jbc.m202960200

Figure Lengend Snippet: FIG. 6. The B-myb promoter can be transcriptionally repressed by transfected p107 (but not Rb) in immortalized p107// p130/ 3T3 cells. A, shown is a Northern blot probed for B-myb expression in 3T3 cells derived from control p107/ and knockout p107//p130/ MEFs. Cells were deprived of serum for 60 h to arrest in G0. Flow cytometry showed that both cell lines were quiescent at this time. The cells were then stimulated by re-addition of serum for 18 h (S phase), by which stage 60% were judged to be in S phase by flow cytometry. B, knockout p107//p130/ 3T3 cells were transfected in triplicate with the wild-type (wt) B-myb promoter/luciferase reporter pGL2-(536) or with variants carrying mutations in the DRS (mDRS) or mutations in both the E2F/DRS sites (mE2F/mDRS) together with a control pCMV plasmid or an expression vector encoding p107 or Myc epitope-tagged Rb and RbNP. Cells were arrested in G0 by serum deprivation for 60 h, and luciferase activities were assayed in cell extracts. The results are presented as -fold repression of transcription by these pocket proteins, where the average luciferase activity obtained with the control empty expression plasmid transfection for each re- porter was given a value of unity. C, EMSA was performed with ex- tracts from knockout p107//p130/ 3T3 cells transfected with the control pCMV vector (CON) or with an expression vector encoding p107, Rb, or RbNP. To detect E2F complexes containing the ectopically ex- pressed pocket proteins, antibody (Ab) supershifts were done with anti- p107 antibody SD15 or antibody 9E10, which recognizes the Myc epitope tag present on the transfected Rb and RbNP proteins.

Article Snippet: These were used in EMSAs as described above; however, the antibodies used in supershifts were the same as those used in ChIP assays (rabbit anti-p107 polyclonal antibody, Santa Cruz Biotechnology sc-318; rabbit anti-p130 polyclonal antibody, Santa Cruz Biotechnology sc-317; and anti-Rb monoclonal antibody 21C9).

Techniques: Transfection, Northern Blot, Expressing, Derivative Assay, Control, Knock-Out, Flow Cytometry, Luciferase, Plasmid Preparation, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: A B-myb Promoter Corepressor Site Facilitatesin Vivo Occupation of the Adjacent E2F Site by p107·E2F and p130·E2F Complexes

doi: 10.1074/jbc.m202960200

Figure Lengend Snippet: FIG. 7. The p130 N terminus is required for transcriptional repression of the B-myb promoter. A, knockout p107//p130/ 3T3 cells were transfected in triplicate with the wild-type B-myb promoter/luciferase reporter pGL2-(536) together with 1- and 2-g amounts as indicated of a control pcDNA3 vector (CON) or pcDNA3 encoding Myc epitope-tagged Rb, p107, or p130. Cells were arrested in G0 by serum deprivation for 60 h, and luciferase activities were assayed in cell extracts. The results are presented as -fold repression by these proteins on the reporter, where the average luciferase activity obtained with the control transfection was given a value of unity. B, Western blotting was carried out using antibody 9E10 as the primary antibody, and the results show the relative levels of expression of Rb, p107, and p130 in transfected p107//p130/ 3T3 cells at inputs of 1 and 2 g of expression vector. Note that p107 appears as a faint band immediately below the major background band obtained in all lanes. The background band is indicated with an asterisk. C, transcriptional repression of the B-myb promoter by p130 requires the E2F binding domain and an N-terminal domain. Knockout p107//p130/ 3T3 cells were transfected in triplicate with the wild-type B-myb promoter/ luciferase reporter pGL2-(536) together with 0.2-, 0.5-, and 1 g-amounts as indicated of a control pcDNA3 vector or pcDNA3 encoding Myc-epitope-tagged p130 and p130 mutants N417 (amino acids 417–1139), C793 (amino acids 2–793), C1065 (amino acids 2–1065), and dl622–818 (deletion of amino acids 622–818 within the spacer). Cells were arrested in G0 by serum deprivation for 60 h, and luciferase activities were assayed in cell extracts. The results are presented as -fold repression by these proteins on the reporter, where the average luciferase activity obtained with the control transfection was given a value of unity. D, Western blotting was performed using antibody 9E10 as the primary antibody, and the results show the relative levels of expression of wild-type and mutant p130 proteins in transfected p107//p130/ 3T3 cells at 1-g input of expression vector. Lane 1, wild-type p130; lane 2, mutant N417; lane 3, mutant C793; lane 4, mutant C1065; lane 5, mutant dl622–818. The major background band is indicated with an asterisk. E, shown is a schematic representation of Myc epitope-tagged p130 and p130 deletion mutants. Myc epitope-tagged p130 contains amino acids 2–1139 of wild-type p130, and the amino acid numbers defining the boundaries of the A and B pockets and the intervening spacer are indicated. Sequences retained in the p130 deletion mutants are indicated below.

Article Snippet: These were used in EMSAs as described above; however, the antibodies used in supershifts were the same as those used in ChIP assays (rabbit anti-p107 polyclonal antibody, Santa Cruz Biotechnology sc-318; rabbit anti-p130 polyclonal antibody, Santa Cruz Biotechnology sc-317; and anti-Rb monoclonal antibody 21C9).

Techniques: Knock-Out, Transfection, Luciferase, Control, Plasmid Preparation, Activity Assay, Western Blot, Expressing, Binding Assay, Mutagenesis

Journal: Journal of Biological Chemistry

Article Title: A B-myb Promoter Corepressor Site Facilitatesin Vivo Occupation of the Adjacent E2F Site by p107·E2F and p130·E2F Complexes

doi: 10.1074/jbc.m202960200

Figure Lengend Snippet: FIG. 8. The B-myb E2F site is unoccupied in p107//p130/

Article Snippet: These were used in EMSAs as described above; however, the antibodies used in supershifts were the same as those used in ChIP assays (rabbit anti-p107 polyclonal antibody, Santa Cruz Biotechnology sc-318; rabbit anti-p130 polyclonal antibody, Santa Cruz Biotechnology sc-317; and anti-Rb monoclonal antibody 21C9).

Techniques: