Journal: Journal of Biological Chemistry

Article Title: UV-mediated Regulation of the Anti-senescence Factor Tbx2

doi: 10.1074/jbc.m705651200

Figure Lengend Snippet: FIGURE 1. Tbx2 is phosphorylated in vivo. A, dephosphorylation of Tbx2 by shrimp alkaline phosphatase. Protein extracts (30 g) from MCF-7 cells were dephosphorylated by incubating the samples with 1 unit of shrimp alkaline phosphatase at 37 °C for 30 min prior to Western blot analyses. For optimal separation of phosphorylated Tbx2, protein extracts were separated by 7.5% SDS-PAGE. Tubulin is included as a loading control. p-Tbx2, phosphorylated Tbx2.B,schematicrepresentationofTbx2showingthethirteenSPmotifsthat are potential phosphorylation sites.

Article Snippet: The primary antibodies used were mouse monoclonal anti-Tbx2 62–2 antibody (1:2000), rabbit polyclonal anti-phospho-p38 (1:1000) and anti-p38 (1:5000) (Cell Signaling Technology Inc., Beverly, MA), anti-Pk-Tag (SV5) antibody (1:3000) (Serotec, Oxford, UK), and rabbit polyclonal p21 (1:200) and mouse monoclonal -tubulin (1:500) from Santa Cruz Biotechnology, Santa Cruz, CA.

Techniques: In Vivo, De-Phosphorylation Assay, Western Blot, SDS Page, Control, Phospho-proteomics

Journal: Journal of Biological Chemistry

Article Title: UV-mediated Regulation of the Anti-senescence Factor Tbx2

doi: 10.1074/jbc.m705651200

Figure Lengend Snippet: FIGURE 2. UVC-induced phosphorylation of Tbx2. A, dose-dependent increase in the phosphorylation status of transfected Tbx2 in response to UVC irradiation. COS-7 cells were transfected with a vector expressing SV5 epitope-tagged Tbx2 and UVC irradiated at the indicated dosages. Protein extracts were prepared 1 h post- treatment. To detect phosphorylated Tbx2, protein samples were analyzed on a 7.5% SDS-PAGE and by West- ern blotting using an antibody to SV5. Levels of phospho-p38 and total p38 in these samples were assessed on a 12% SDS-PAGE and by Western blotting with appropriate antibodies. The bar graph compares the intensity oftheupperandlowerbandsineachsamplenormalizedtothep38loadingcontrol.B,time-dependentincrease inthephosphorylationofendogenousTbx2inresponsetoUVCirradiation.ProteinextractsfromMCF-7cellsat the indicated time points post-UVC irradiation (100 J/m2) were assayed as described in panel A. The proteins extracted from 0 to 30 min and 60 to 300 min post-UVC treatment were from different experiments and were therefore analyzed on separate gels. C, endogenous Tbx2 is phosphorylated in vivo by the p38 kinase. MCF-7 cells irradiated with UVC (100 J/m2) in the presence or absence of SB203580 (10 M, 30 min prior to UVC irradiation), a highly specific inhibitor of the p38 family kinases. Protein extracts were assayed as described in panelA.D,Tbx2mRNAlevelsincreaseinresponsetoUVCirradiation.Quantitativereal-timePCRofendogenous Tbx2 mRNA extracted from MCF-7 cells was performed at the indicated times post-UVC irradiation (100 J/m2). The levels of Tbx2 mRNA expression were normalized against glyceraldehyde-3-phosphate dehydrogenase. Error bars represent standard deviations.

Article Snippet: The primary antibodies used were mouse monoclonal anti-Tbx2 62–2 antibody (1:2000), rabbit polyclonal anti-phospho-p38 (1:1000) and anti-p38 (1:5000) (Cell Signaling Technology Inc., Beverly, MA), anti-Pk-Tag (SV5) antibody (1:3000) (Serotec, Oxford, UK), and rabbit polyclonal p21 (1:200) and mouse monoclonal -tubulin (1:500) from Santa Cruz Biotechnology, Santa Cruz, CA.

Techniques: Phospho-proteomics, Transfection, Irradiation, Plasmid Preparation, Expressing, SDS Page, Western Blot, In Vivo

Journal: Journal of Biological Chemistry

Article Title: UV-mediated Regulation of the Anti-senescence Factor Tbx2

doi: 10.1074/jbc.m705651200

Figure Lengend Snippet: FIGURE 3. Tbx2 is phosphorylated by the p38 MAP kinase at three serine residues. A, schematic represen- tation of the N-terminal (1–371) and C-terminal (371–711) Tbx2 proteins used as substrates in p38 kinase assays.B,mappingthep38MAPkinasetargetsiteswithinTbx2.Invitrop38kinaseassayswereperformedusing purified GST-Tbx2 fusion proteins as substrates in the presence of the recombinant activated p38 kinase and [-32P]ATP. Kinase assays using the indicated Tbx2 proteins are shown in the upper panels after SDS-PAGE and autoradiography.ThelowerpanelsshowthesamegelsstainedwithCoomassieBlue,indicatingthatequivalent amounts of protein were used in each assay. The bar graph shows densitometric values of radioactive levels measured for each construct in the kinase assay. C, mutating the p38 MAP kinase sites affects the phosphoryl- ation of Tbx2 in vivo. SV5 epitope-tagged Tbx2 or the S336A,S623A,S675A mutant were expressed in COS-7 cells, and the phosphorylation status of Tbx2 was analyzed by 7.5% SDS-PAGE and by Western blotting using an anti-SV5 antibody. D, the identified p38 target serine residues 336, 623, and 675 in the Tbx2 protein are the only sites phosphorylated in response to UVC irradiation. Cells were transfected with SV5 epitope- tagged WT Tbx2 or the Tbx2 S336A,S623A,S675A mutant and exposed to UVC irradiation (40 J/m2) 29 h post-transfection. Protein extracts were prepared 1 h post-UVC treatment and analyzed by Western blot- ting as described in Fig. 2A.

Article Snippet: The primary antibodies used were mouse monoclonal anti-Tbx2 62–2 antibody (1:2000), rabbit polyclonal anti-phospho-p38 (1:1000) and anti-p38 (1:5000) (Cell Signaling Technology Inc., Beverly, MA), anti-Pk-Tag (SV5) antibody (1:3000) (Serotec, Oxford, UK), and rabbit polyclonal p21 (1:200) and mouse monoclonal -tubulin (1:500) from Santa Cruz Biotechnology, Santa Cruz, CA.

Techniques: Purification, Recombinant, SDS Page, Autoradiography, Construct, Kinase Assay, In Vivo, Mutagenesis, Phospho-proteomics, Western Blot, Irradiation, Transfection

Journal: Journal of Biological Chemistry

Article Title: UV-mediated Regulation of the Anti-senescence Factor Tbx2

doi: 10.1074/jbc.m705651200

Figure Lengend Snippet: FIGURE 4. UVC-induced phosphorylation by p38 affects Tbx2 protein stability and subcellular localization. A, the Tbx2 S336A,S623A,S675A mutant displays a reduced half-life compared with wild-type Tbx2 and the Tbx2 S336E,S623E,S675E mutant. NIH 3T3 cells, transiently transfected with vectors expressingSV5epitope-taggedTbx2proteinsasindicated,wereincubated48hpost-transfectionwith30g/mlcycloheximidefortheindicatedtimestoblock de novo protein synthesis. To accurately detect total levels of the Tbx2 protein as a single band, cell lysates were analyzed on a 10% SDS-PAGE and by Western blotting with anti-SV5 antibodies. The bar graph compares the intensity of the Tbx2 protein normalized to the loading control. B, p38 phosphorylation induces nuclear translocation of Tbx2. MCF-7 cells were treated with the p38 inhibitor SB203580 prior to UVC irradiation (100 J/m2) and then analyzed 1 h post- treatment by immunofluorescence using an anti-Tbx2 antibody. The upper and lower panels represent images captured at 40 and 100 magnifications, respectively.

Article Snippet: The primary antibodies used were mouse monoclonal anti-Tbx2 62–2 antibody (1:2000), rabbit polyclonal anti-phospho-p38 (1:1000) and anti-p38 (1:5000) (Cell Signaling Technology Inc., Beverly, MA), anti-Pk-Tag (SV5) antibody (1:3000) (Serotec, Oxford, UK), and rabbit polyclonal p21 (1:200) and mouse monoclonal -tubulin (1:500) from Santa Cruz Biotechnology, Santa Cruz, CA.

Techniques: Phospho-proteomics, Mutagenesis, Transfection, SDS Page, Western Blot, Control, Translocation Assay, Irradiation, Immunofluorescence

Journal: Journal of Biological Chemistry

Article Title: UV-mediated Regulation of the Anti-senescence Factor Tbx2

doi: 10.1074/jbc.m705651200

Figure Lengend Snippet: FIGURE 5. UVC-induced phosphorylation by p38 enhances the ability of Tbx2 to repress p21. A, inverse correlation between Tbx2 and p21 protein levels in response to UVC irradiation. Protein extracts (the same used in Fig. 2B) from MCF-7 cells at the indicated time points post-UVC irradiation (100 J/m2) were analyzed by 15% SDS-PAGE and Western blotting using an antibody to p21. The proteins extracted from 0 to 30 min and 60 to 300 min post-UVC treatment were analyzed on different gels. The results for Tbx2, p-p38, and total p38 are the same as shown in Fig. 2B. The bar graph compares the intensity of the Tbx2, p-p38, and p21 bands in each sample normalized to the p38 loading control. B, p21 mRNA levels are repressed in response to UVC irradiation. Total RNA was extracted from MCF-7 cells at the times indicated post-UVC irradiation. Quantitative real-time PCR was then performed on reverse-transcribed RNA using primersspecifictop21,andmRNAlevelswerenormalizedtoglyceraldehyde-3-phosphatedehydrogenase.Errorbarsrepresentstandarddeviations.C,UVCirradiation enhancesthetranscriptionalrepressionofp21byTbx2.Thep21promoter-luciferasereporter(700ng)wasco-transfectedintoCOS-7cellseitherwiththeemptypCMV (200 ng) vector or a Tbx2 (200 ng) expression vector in the presence or absence of UVC (40 J/m2) and luciferase activity determined. Promoter activity is indicated as -fold repression, which represents the ratio of the luciferase activity generated by the pCMV empty vector (without Tbx2) to that obtained in the presence of pCMV-Tbx2.Theaboveexperimentwasrepeatedunderthesameconditionsusing700ngofthep14ARFpromoter-luciferasereporter.*,significantdifferencefromthe control (no UVC treatment) at p 0.05. D, COS-7 cells were transfected as described in panel C with the Tbx2 expression vectors as indicated and luciferase activity determined. Western blotting shows equal expression of WT Tbx2 and the Tbx2 mutant in the luciferase assays. Error bars represent standard deviations.

Article Snippet: The primary antibodies used were mouse monoclonal anti-Tbx2 62–2 antibody (1:2000), rabbit polyclonal anti-phospho-p38 (1:1000) and anti-p38 (1:5000) (Cell Signaling Technology Inc., Beverly, MA), anti-Pk-Tag (SV5) antibody (1:3000) (Serotec, Oxford, UK), and rabbit polyclonal p21 (1:200) and mouse monoclonal -tubulin (1:500) from Santa Cruz Biotechnology, Santa Cruz, CA.

Techniques: Phospho-proteomics, Irradiation, SDS Page, Western Blot, Control, Real-time Polymerase Chain Reaction, Reverse Transcription, Plasmid Preparation, Expressing, Luciferase, Activity Assay, Generated, Transfection, Mutagenesis