Journal: PLoS ONE

Article Title: Regulation of Eukaryotic Initiation Factor 4AII by MyoD during Murine Myogenic Cell Differentiation

doi: 10.1371/journal.pone.0087237

Figure Lengend Snippet: ( A ) Schematic representation of the eIF4A and eIF4AII promoters showing the relative position and nucleotide sequence of putative MyoD binding sites. Positions are relative to the transcription start site (+1). Arrows denote the relative position of the primers used in the ChIP assay. ( B ) ChIP assays performed with C2C12 extracts prepared on the indicated days following induction of differentiation. Equivalent amounts of crosslinked chromatin were immunoprecipitated using either an anti-MyoD antibody or an IgG control. The presence of eIF4AI and eIF4AII promoter sequences in the immunoprecipitations was evaluated by qPCR. Primers to the myogenin promoter were used as control and values are normalized to input levels. The input sample represents 5% of the initial DNA material following sonication before immunoprecipitation. n = 4±SEM. ( C ) Products of qPCRs from ChIP assays performed in (B) (In-Input, Ig- IgG elution, MD- MyoD elution).

Article Snippet: Immunoprecipitations were performed with either anti-MyoD antibody (M-318, sc-760, Santa Cruz Biotech) or IgG control at 4°C overnight with tubes rotating end-over-end.

Techniques: Sequencing, Binding Assay, Immunoprecipitation, Control, Sonication

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: Human molecular genetics

Article Title: The product of an oculopharyngeal muscular dystrophy gene, poly(A)-binding protein 2, interacts with SKIP and stimulates muscle-specific gene expression.

doi: 10.1093/hmg/10.11.1129

Figure Lengend Snippet: Figure 1. Enhanced myotube formation in stable C2 cells expressing human PABP2. Phase contrast and immunofluorescence images were taken from the cells cultured in growth medium (GM, day 0) and 2 and 3 days in differentiation medium (DM, day 2 and day 3). Anti-Flag polyclonal antibody was used to detect human Flag-PABP2. Scale bar, 50 µm.

Article Snippet: For immunoprecipitation the diluted cell lysate was incubated with 2 μg of anti-Flag polyclonal antibody or anti-MyoD polyclonal antibody (M-318; Santa Cruz Biotechnology) and 30 μl of protein G–Sepharose for 2 h at 4°C with slow rotation.

Techniques: Expressing, Immunofluorescence, Cell Culture

Journal: Human molecular genetics

Article Title: The product of an oculopharyngeal muscular dystrophy gene, poly(A)-binding protein 2, interacts with SKIP and stimulates muscle-specific gene expression.

doi: 10.1093/hmg/10.11.1129

Figure Lengend Snippet: Figure 3. The effect of PABP2 transfection on MyoD gene transcription. C2 cells were transiently transfected with 8 µg of PABP2 expression plasmid or control vector plasmid in a 100 mm culture dish. Twenty-four hours after transfection the culture medium was changed to a differentiation medium. After an additional 48 h incubation, cells were harvested for nuclear run-on assay (A) and northern blot analysis (B).

Article Snippet: For immunoprecipitation the diluted cell lysate was incubated with 2 μg of anti-Flag polyclonal antibody or anti-MyoD polyclonal antibody (M-318; Santa Cruz Biotechnology) and 30 μl of protein G–Sepharose for 2 h at 4°C with slow rotation.

Techniques: Transfection, Expressing, Plasmid Preparation, Control, Incubation, Nuclear Run-on Assay, Northern Blot

Journal: Human molecular genetics

Article Title: The product of an oculopharyngeal muscular dystrophy gene, poly(A)-binding protein 2, interacts with SKIP and stimulates muscle-specific gene expression.

doi: 10.1093/hmg/10.11.1129

Figure Lengend Snippet: Figure 4. The N-terminal region of PABP2 is responsible for the up-regulation of MyoD and myogenin mRNAs. (A) Structures of the full-length and truncated PABP2 cDNA used in this experiment. (B) The protein expressions in the C2 cells transiently transfected with indicated expression plasmids were verified by western blot analysis using anti-Flag polyclonal antibody. Cells were directly dissolved in SDS-sample buffer and subjected to western blot analysis. (C) Quantitative RT–PCR resulting from C2 cells transiently transfected with 2 µg of expression plasmids encoding full-length or truncated PABP2 cDNA is shown. Approximately 5 × 105 cells were plated on 60 mm dishes in growth medium 24 h prior to transfection. The cells almost reached confluence 24 h after transfection. At this time, the growth medium was replaced with a differentiation medium. The ratio of target cDNA/β-actin cDNA amounts from the mock controls at 24 h after transfection was arbitrarily set at 1. The values are derived from two sets of independently transfected triplicates. Error bars indicate the SD of the means.

Article Snippet: For immunoprecipitation the diluted cell lysate was incubated with 2 μg of anti-Flag polyclonal antibody or anti-MyoD polyclonal antibody (M-318; Santa Cruz Biotechnology) and 30 μl of protein G–Sepharose for 2 h at 4°C with slow rotation.

Techniques: Transfection, Expressing, Western Blot, Quantitative RT-PCR, Derivative Assay

Journal: Human molecular genetics

Article Title: The product of an oculopharyngeal muscular dystrophy gene, poly(A)-binding protein 2, interacts with SKIP and stimulates muscle-specific gene expression.

doi: 10.1093/hmg/10.11.1129

Figure Lengend Snippet: Figure 5. Interaction of PABP2 and SKIP. (A) GST-pulldown assay shows the interaction between GST-PABP2 and Myc-SKIP. To construct the expression plasmids for GST-fusion proteins, previously described PABP2 cDNA fragments (Fig. 4A) were used. The indicated fusion proteins were expressed in E.coli (BL21), purified, and then incubated with the lysate from COS-7 cells transfected with the Myc-SKIP expression plasmid. The associated proteins were recovered with glutathione-Sepharose resin, and then subjected to western blot (WB) analysis using anti-Myc monoclonal antibody. Five percent of cell lysates were loaded as the Input. (B) Myc-SKIP is co-immunoprecipitated with Flag-PABP2. Cell lysates from COS-7 transfected with the indicated expression plasmids were immunoprecipitated using the anti-Flag polyclonal antibody. Immunoprecipitates (IP) were analyzed by western blot using anti-Myc monoclonal antibody for Myc-SKIP and anti-Flag M2 monoclonal antibody for Flag-PABP2. The arrowhead indicates the immunoglobulin heavy chain. Figure 6. Nuclear localization of PABP2 and SKIP. HeLa cells were transfected with the expression plasmids for Flag-PABP2 and/or Myc-SKIP. A single confocal scan is shown. Scale bar, 5 µm. (A) Flag-PABP2 (green) localized in nuclear speckles (top). Cells were counter-stained with endogenous SC-35 (red). Co-transfected HeLa cells (bottom) show that Flag-PABP2 (green) is co-localized with Myc-SKIP in the nuclear speckles (red). (B) Intranuclear aggregates formation in HeLa cells transiently transfected with Flag-PABP2 expression plasmid. Approximately 5% of Flag-PABP2 transiently transfected cells show the aggregate bodies at 48 h after transfection. The nuclear distribution of SC-35 and Myc-SKIP were changed and were strongly associated with aggregates by PABP2 expression.

Article Snippet: For immunoprecipitation the diluted cell lysate was incubated with 2 μg of anti-Flag polyclonal antibody or anti-MyoD polyclonal antibody (M-318; Santa Cruz Biotechnology) and 30 μl of protein G–Sepharose for 2 h at 4°C with slow rotation.

Techniques: GST Pulldown Assay, Construct, Expressing, Purification, Incubation, Transfection, Plasmid Preparation, Western Blot, Immunoprecipitation, Staining

Journal: Human molecular genetics

Article Title: The product of an oculopharyngeal muscular dystrophy gene, poly(A)-binding protein 2, interacts with SKIP and stimulates muscle-specific gene expression.

doi: 10.1093/hmg/10.11.1129

Figure Lengend Snippet: Figure 7. PABP2 co-operates with SKIP to stimulate E-box-mediated transcription through MyoD. (A) C3H10T1/2 cells were transiently transfected with reporter plasmid (4RE-tk-luc) and expression plasmids encoding PABP2, SKIP and MyoD as indicated. The increasing amounts (0.2, 0.4, 0.6 and 0.8 µg, respectively) of expression plasmids for PABP2 and SKIP were used for trans- fection. (B) The expression plasmids encoding full-length or truncated forms of PABP2 were used as described in Figure 4A. The amount of PABP2 and SKIP expression plasmids used for transfections was 0.4 and 0.2 µg, respectively. The total amount of DNA in each transfection was kept constant by addition of pUC 18 DNA. The reporter activity from the cells transfected with reporter plasmid alone was assigned a value of 1. The values are derived from two sets of independently transfected triplicates. Error bars indicate the SD of the means.

Article Snippet: For immunoprecipitation the diluted cell lysate was incubated with 2 μg of anti-Flag polyclonal antibody or anti-MyoD polyclonal antibody (M-318; Santa Cruz Biotechnology) and 30 μl of protein G–Sepharose for 2 h at 4°C with slow rotation.

Techniques: Transfection, Plasmid Preparation, Expressing, Activity Assay, Derivative Assay

Journal: Human molecular genetics

Article Title: The product of an oculopharyngeal muscular dystrophy gene, poly(A)-binding protein 2, interacts with SKIP and stimulates muscle-specific gene expression.

doi: 10.1093/hmg/10.11.1129

Figure Lengend Snippet: Figure 8. PABP2 and SKIP associate with MyoD. C3H10T1/2 cells were transiently transfected with 1 µg of MyoD and SKIP expression plasmids and 2 µg of PABP2 expression plasmid in 60 mm culture plates. The cell lysates were immunoprecipitated using anti-MyoD polyclonal antibody. Immunoprecipitates (IP) were analyzed by western blotting using anti-Myc monoclonal antibody, anti-Flag monoclonal antibody (M2) and anti-MyoD monoclonal antibody. The arrowheads indicate the immunoglobulin heavy chains.

Article Snippet: For immunoprecipitation the diluted cell lysate was incubated with 2 μg of anti-Flag polyclonal antibody or anti-MyoD polyclonal antibody (M-318; Santa Cruz Biotechnology) and 30 μl of protein G–Sepharose for 2 h at 4°C with slow rotation.

Techniques: Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot