Journal:

Article Title: Transient alteration of T cell fine specificity by a strong primary stimulus correlates with T cell receptor down-regulation

doi:

Figure Lengend Snippet: Inhibition of protein tyrosine kinase activity blocks TCR down-regulation. P14 TCR transgenic spleen cells (A, B) or P14.G5 CTL clone (C, D) were preincubated for 60 min in the presence of PP1 or genistein. Preincubation with herbimycin A was done overnight with P14 spleen cells or for 60 min with P14.G5 CTL. EL-4 cells pulsed with p33 (1 μM) were then added for 4 h and cells stained for flow cytometry with anti-CD8 and anti-Vα2 antibodies (A, C) or with anti-CD8 and anti-CD69 antibodies (B, D). Mean fluorescence intensities are expressed as % or MFI of control in the absence of peptide.

Article Snippet: P14.G5 CTL (6×10 4 /well) or P14 spleen cells (5×10 5 /well) were preincubated for 60 min with the src family kinase-specific inhibitor PP1 (10 μM and 50 μM, respectively) (Bio-mol, Plymouth Meeting, PA) [ 28 ].

Techniques: Inhibition, Activity Assay, Transgenic Assay, Staining, Flow Cytometry, Fluorescence, Control

Journal: The Journal of Biological Chemistry

Article Title: α6 Integrin Transactivates Insulin-like Growth Factor Receptor-1 (IGF-1R) to Regulate Caspase-3-mediated Lens Epithelial Cell Differentiation Initiation *

doi: 10.1074/jbc.M113.515254

Figure Lengend Snippet: Activation of IGF-1R in α6 integrin signaling complexes is dependent on the kinase activity of Src family kinases. Primary lens cells in culture were exposed to the highly specific Src family kinase inhibitor, PP1, or to the vehicle dimethyl sulfoxide (DMSO) for 4 h. Following PP1 treatment, cells were immunoprecipitated for α6 integrin and the immunoprecipitates were immunoblotted for α6 integrin and phospho-Src (p-Src) (A), Src (B), phospho-IGF-1R (p-IGF-1R) (C), and IGF-1R (D). A–D, graphical representation of densitometric analysis of co-immunoprecipitation studies showing the ratio of activated, p-Src/α6 integrin (A), total Src/α6 integrin (B), activated p-IGF-1R/α6 integrin (C), and total IGF-1R/α6 integrin (D). Data shows that blocking activation of Src kinases prevented α6 integrin transactivation of IGF-1R. Results shown are quantified from more than three independent studies. Error bars represent S.E. *, p < 0.05, t test.

Article Snippet: For blocking activation of Src family kinases, cells were exposed to the Src family kinase-specific inhibitor PP1 (10 μ m , Enzo Life Sciences, Farmingdale, NY) for 4 h. Controls were treated with the vehicle dimethyl sulfoxide.

Techniques: Activation Assay, Activity Assay, Immunoprecipitation, Blocking Assay

Journal: Molecular and Cellular Biology

Article Title: Oxidized DJ-1 Inhibits p53 by Sequestering p53 from Promoters in a DNA-Binding Affinity-Dependent Manner

doi: 10.1128/MCB.01350-12

Figure Lengend Snippet: DJ-1–p53 complex stably binds to the p21 promoter but not to the DUSP1 promoter. H1299 cells were transfected with FLAG-p53 and wild-type or C106S DJ-1–HA. Forty-eight hours after transfection, cells were treated with 300 μM H2O2 for 30 min, and ChIP assays targeting the DUSP1 (A) and p21 (B) genes were carried out. (C) Proteins prepared from transfected H1299 cells were analyzed by Western blotting. A549 cells were treated with 300 μM H2O2 for 30 min, and ChIP assays targeting the DUSP1 (D) and p21 (E) genes were carried out. The intensities of the precipitated DNA bands in lanes 4 and 6 in panel E were quantified, and their relative levels are shown. (F) Proteins prepared from H2O2-treated A549 cells were analyzed by Western blotting. Ab, antibody.

Article Snippet: The antibodies used in this study were anti-HA (1:2,000) (MBL, Nagoya, Japan), anti-FLAG F7425 (1:1,000) (Sigma), anti-T7 (1:1,000) (Novagen, Madison, WI), anti-p53 (DO-1 at 1:1,000) (Santa Cruz Biotechnology, Santa Cruz, CA), anti-phospho-p53(serine 6) (1:1,000) (Cell Signaling Technology, Danvers, MA), anti-phospho-p53(serine 9) (1:1,000) (Cell Signaling Technology), anti-phospho-p53(serine 15) (1:1,000) (Cell Signaling Technology), anti-phospho-p53(serine 20) (1:1,000) (Cell Signaling Technology), anti-phospho-p53(serine 37) (1:1,000) (Cell Signaling Technology), anti-phospho-p53(serine 46) (1:1,000) (Cell Signaling Technology), anti-phospho-p53(serine 392) (1:1,000) (Cell Signaling Technology), anti-actin (1:4,000) (Chemicon, Temecula, CA), anti-phospho-ERK1/2 (1:1,000) (Cell Signaling Technology), anti-ERK1/2 (1:1,000) (Santa Cruz Biotechnology), anti-p53 (Pab 240 at 1:1,000) (Santa Cruz Biotechnology), anti-mitogen-activated protein kinase (MAPK) phosphatase 1 (MKP1) (C-19 at 1:500) (Santa Cruz Biotechnology), monoclonal rat anti-DJ-1 (1:1,000), polyclonal anti-DJ-1 (1:4,000), monoclonal mouse anti-DJ-1 (3E8 at 1: 4,000) (MBL), and anti-oxidized DJ-1 (1:1,000).

Techniques: Stable Transfection, Transfection, Western Blot

Journal: Molecular and Cellular Biology

Article Title: Oxidized DJ-1 Inhibits p53 by Sequestering p53 from Promoters in a DNA-Binding Affinity-Dependent Manner

doi: 10.1128/MCB.01350-12

Figure Lengend Snippet: A time course of endogenous DJ-1–p53 interactions and endogenous p53 ChIP on the DUSP promoter in cells exposed to oxidative stress. (A) A549 cells were treated with 300 μM H2O2 for 0.25 to 2 h, and ChIP assays targeting the DUSP1 gene were carried out. (B) Proteins prepared from H2O2-treated A549 cells were analyzed by Western blotting. (C) DJ-1+/+ and DJ-1−/− mouse cells were treated with 300 μM H2O2 for 0.5 h, and ChIP assays targeting the DUSP1 gene were carried out. (D) Proteins prepared from H2O2-treated mouse DJ-1+/+ and DJ-1−/− cells were analyzed by Western blotting.

Article Snippet: The antibodies used in this study were anti-HA (1:2,000) (MBL, Nagoya, Japan), anti-FLAG F7425 (1:1,000) (Sigma), anti-T7 (1:1,000) (Novagen, Madison, WI), anti-p53 (DO-1 at 1:1,000) (Santa Cruz Biotechnology, Santa Cruz, CA), anti-phospho-p53(serine 6) (1:1,000) (Cell Signaling Technology, Danvers, MA), anti-phospho-p53(serine 9) (1:1,000) (Cell Signaling Technology), anti-phospho-p53(serine 15) (1:1,000) (Cell Signaling Technology), anti-phospho-p53(serine 20) (1:1,000) (Cell Signaling Technology), anti-phospho-p53(serine 37) (1:1,000) (Cell Signaling Technology), anti-phospho-p53(serine 46) (1:1,000) (Cell Signaling Technology), anti-phospho-p53(serine 392) (1:1,000) (Cell Signaling Technology), anti-actin (1:4,000) (Chemicon, Temecula, CA), anti-phospho-ERK1/2 (1:1,000) (Cell Signaling Technology), anti-ERK1/2 (1:1,000) (Santa Cruz Biotechnology), anti-p53 (Pab 240 at 1:1,000) (Santa Cruz Biotechnology), anti-mitogen-activated protein kinase (MAPK) phosphatase 1 (MKP1) (C-19 at 1:500) (Santa Cruz Biotechnology), monoclonal rat anti-DJ-1 (1:1,000), polyclonal anti-DJ-1 (1:4,000), monoclonal mouse anti-DJ-1 (3E8 at 1: 4,000) (MBL), and anti-oxidized DJ-1 (1:1,000).

Techniques: Western Blot

Journal: Molecular and Cellular Biology

Article Title: Oxidized DJ-1 Inhibits p53 by Sequestering p53 from Promoters in a DNA-Binding Affinity-Dependent Manner

doi: 10.1128/MCB.01350-12

Figure Lengend Snippet: DJ-1 downregulates DUSP1 expression under an oxidative stress condition. (A) Mouse primary cells were treated with H2O2 for 0.25 to 6 h; the expression levels of respective mRNAs were examined by semiquantitative RT-PCR, and their expression levels relative to that of β-actin are shown. (B) HEK293T cells were transfected with FLAG-p53 and DJ-1–HA and treated with 1 mM H2O2 for 15 to 45 min (left panel) or 0.5 to 4 h (right panel) at 48 h after transfection. Proteins were analyzed by immunoprecipitation followed by Western blotting. (C) A549 cells were treated with 300 μM H2O2 for 15 to 120 min. Proteins were analyzed by immunoprecipitation with an anti-DJ-1 antibody followed by Western blotting. (D and E) DJ-1+/+ and DJ-1−/− mouse cells were treated with H2O2 for 0.5 h or 2 h. The expression levels of DUSP1 (D) and p21 mRNA (E) were examined by semiquantitative RT-PCR, and their expression levels relative to that of β-actin are shown. (F and G) DJ-1+/+ and DJ-1−/− mouse cells were treated with H2O2 for 0.5 and 2 h. The expression levels of DUSP1 (F) and p21 mRNA (G) were examined by quantitative RT-PCR. (H) DJ-1+/+ and DJ-1−/− mouse cells were treated with H2O2 for 0.5 and 2 h. The expression levels of DUSP1, p21, p53, DJ-1, and β-actin were analyzed by Western blotting. (I) DJ-1+/+ and DJ-1−/− mouse cells were transfected with control siRNA or p53 siRNA and treated with 300 μM H2O2 for 30 min or 2 h at 72 h after transfection. The expression levels of DUSP1 and p21 mRNAs were examined by semiquantitative RT-PCR. The nucleotide sequences of the p53 siRNA are 5′-CCAGAAGAUAUCCUGCCAUTT-3′ (mp53 sense) and 5′-AUGGCAGGAUAUCUUCUGGTT-3′ (mp53 antisense). (J and K) DJ-1+/+ and DJ-1−/− mouse cells were treated with H2O2 for 0.5 and 2 h. p53 was visualized as described in Materials and Methods. The values shown in panels D through G are means ± SE (n = 3 experiments). **, P < 0.01; ***, P < 0.001. N.S. indicates no significance.

Article Snippet: The antibodies used in this study were anti-HA (1:2,000) (MBL, Nagoya, Japan), anti-FLAG F7425 (1:1,000) (Sigma), anti-T7 (1:1,000) (Novagen, Madison, WI), anti-p53 (DO-1 at 1:1,000) (Santa Cruz Biotechnology, Santa Cruz, CA), anti-phospho-p53(serine 6) (1:1,000) (Cell Signaling Technology, Danvers, MA), anti-phospho-p53(serine 9) (1:1,000) (Cell Signaling Technology), anti-phospho-p53(serine 15) (1:1,000) (Cell Signaling Technology), anti-phospho-p53(serine 20) (1:1,000) (Cell Signaling Technology), anti-phospho-p53(serine 37) (1:1,000) (Cell Signaling Technology), anti-phospho-p53(serine 46) (1:1,000) (Cell Signaling Technology), anti-phospho-p53(serine 392) (1:1,000) (Cell Signaling Technology), anti-actin (1:4,000) (Chemicon, Temecula, CA), anti-phospho-ERK1/2 (1:1,000) (Cell Signaling Technology), anti-ERK1/2 (1:1,000) (Santa Cruz Biotechnology), anti-p53 (Pab 240 at 1:1,000) (Santa Cruz Biotechnology), anti-mitogen-activated protein kinase (MAPK) phosphatase 1 (MKP1) (C-19 at 1:500) (Santa Cruz Biotechnology), monoclonal rat anti-DJ-1 (1:1,000), polyclonal anti-DJ-1 (1:4,000), monoclonal mouse anti-DJ-1 (3E8 at 1: 4,000) (MBL), and anti-oxidized DJ-1 (1:1,000).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection, Immunoprecipitation, Western Blot, Quantitative RT-PCR

Journal: Molecular and Cellular Biology

Article Title: Oxidized DJ-1 Inhibits p53 by Sequestering p53 from Promoters in a DNA-Binding Affinity-Dependent Manner

doi: 10.1128/MCB.01350-12

Figure Lengend Snippet: Expression of DUSP1, p21, and NOXA in UV- and doxorubicin-treated cells. Mouse DJ-1+/+ cells were treated with 20 J/m2 UV irradiation (A and C) or 1 μM doxorubicin (B an D) for 0.5 to 6 h. The expression levels of mRNA and protein of DUSP1, p21, and NOXA were examined by semiquantitative RT-PCR (A and B) and by Western blotting (C and D), respectively.

Article Snippet: The antibodies used in this study were anti-HA (1:2,000) (MBL, Nagoya, Japan), anti-FLAG F7425 (1:1,000) (Sigma), anti-T7 (1:1,000) (Novagen, Madison, WI), anti-p53 (DO-1 at 1:1,000) (Santa Cruz Biotechnology, Santa Cruz, CA), anti-phospho-p53(serine 6) (1:1,000) (Cell Signaling Technology, Danvers, MA), anti-phospho-p53(serine 9) (1:1,000) (Cell Signaling Technology), anti-phospho-p53(serine 15) (1:1,000) (Cell Signaling Technology), anti-phospho-p53(serine 20) (1:1,000) (Cell Signaling Technology), anti-phospho-p53(serine 37) (1:1,000) (Cell Signaling Technology), anti-phospho-p53(serine 46) (1:1,000) (Cell Signaling Technology), anti-phospho-p53(serine 392) (1:1,000) (Cell Signaling Technology), anti-actin (1:4,000) (Chemicon, Temecula, CA), anti-phospho-ERK1/2 (1:1,000) (Cell Signaling Technology), anti-ERK1/2 (1:1,000) (Santa Cruz Biotechnology), anti-p53 (Pab 240 at 1:1,000) (Santa Cruz Biotechnology), anti-mitogen-activated protein kinase (MAPK) phosphatase 1 (MKP1) (C-19 at 1:500) (Santa Cruz Biotechnology), monoclonal rat anti-DJ-1 (1:1,000), polyclonal anti-DJ-1 (1:4,000), monoclonal mouse anti-DJ-1 (3E8 at 1: 4,000) (MBL), and anti-oxidized DJ-1 (1:1,000).

Techniques: Expressing, Irradiation, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Molecular and Cellular Biology

Article Title: Oxidized DJ-1 Inhibits p53 by Sequestering p53 from Promoters in a DNA-Binding Affinity-Dependent Manner

doi: 10.1128/MCB.01350-12

Figure Lengend Snippet: Raw data from three sets of luciferase assays. H1299 cells were cotransfected with pGL4.12-DUSP1-luciferase (A) or pGL4.12-p21-luciferase (B) and expression vectors for FLAG-p53 and wild-type or C106S DJ-1–HA. Twenty-four hours after transfection, cells were treated with 300 μM H2O2 or with 10 μg/ml cycloheximide (CHX) for 30 min. Luciferase activities were then calculated. (C and D) Proteins were prepared from H1299 cells transfected with FLAG-p53 and wild-type or C106S DJ-1–HA and analyzed by Western blotting.

Article Snippet: The antibodies used in this study were anti-HA (1:2,000) (MBL, Nagoya, Japan), anti-FLAG F7425 (1:1,000) (Sigma), anti-T7 (1:1,000) (Novagen, Madison, WI), anti-p53 (DO-1 at 1:1,000) (Santa Cruz Biotechnology, Santa Cruz, CA), anti-phospho-p53(serine 6) (1:1,000) (Cell Signaling Technology, Danvers, MA), anti-phospho-p53(serine 9) (1:1,000) (Cell Signaling Technology), anti-phospho-p53(serine 15) (1:1,000) (Cell Signaling Technology), anti-phospho-p53(serine 20) (1:1,000) (Cell Signaling Technology), anti-phospho-p53(serine 37) (1:1,000) (Cell Signaling Technology), anti-phospho-p53(serine 46) (1:1,000) (Cell Signaling Technology), anti-phospho-p53(serine 392) (1:1,000) (Cell Signaling Technology), anti-actin (1:4,000) (Chemicon, Temecula, CA), anti-phospho-ERK1/2 (1:1,000) (Cell Signaling Technology), anti-ERK1/2 (1:1,000) (Santa Cruz Biotechnology), anti-p53 (Pab 240 at 1:1,000) (Santa Cruz Biotechnology), anti-mitogen-activated protein kinase (MAPK) phosphatase 1 (MKP1) (C-19 at 1:500) (Santa Cruz Biotechnology), monoclonal rat anti-DJ-1 (1:1,000), polyclonal anti-DJ-1 (1:4,000), monoclonal mouse anti-DJ-1 (3E8 at 1: 4,000) (MBL), and anti-oxidized DJ-1 (1:1,000).

Techniques: Luciferase, Expressing, Transfection, Western Blot

Journal: Molecular and Cellular Biology

Article Title: Oxidized DJ-1 Inhibits p53 by Sequestering p53 from Promoters in a DNA-Binding Affinity-Dependent Manner

doi: 10.1128/MCB.01350-12

Figure Lengend Snippet: Cysteine 106 of DJ-1 is essential for the repression of p53-dependent DUSP1 transcription under conditions of oxidative stress. H1299 cells were cotransfected with pGL4.12-DUSP1-luciferase (A), pGL4.12-p21-luciferase (B), or pGL4.12-luciferase (C) and FLAG-p53 and wild-type or C106S DJ-1–HA. Cells were treated as described for Fig. 6. The fold repression values of luciferase activities were calculated as described in the text. (D) H1299 cells were transfected with FLAG-p53 and DJ-1–HA and treated with H2O2 for 30 min in the absence of cycloheximide as described for Fig. 6. The expression levels of DUSP1 mRNA were examined by quantitative RT-PCR. Values are means ± SE (n = 3 experiments). **, P < 0.01; ***, P < 0.001. N.S. indicates no significance.

Article Snippet: The antibodies used in this study were anti-HA (1:2,000) (MBL, Nagoya, Japan), anti-FLAG F7425 (1:1,000) (Sigma), anti-T7 (1:1,000) (Novagen, Madison, WI), anti-p53 (DO-1 at 1:1,000) (Santa Cruz Biotechnology, Santa Cruz, CA), anti-phospho-p53(serine 6) (1:1,000) (Cell Signaling Technology, Danvers, MA), anti-phospho-p53(serine 9) (1:1,000) (Cell Signaling Technology), anti-phospho-p53(serine 15) (1:1,000) (Cell Signaling Technology), anti-phospho-p53(serine 20) (1:1,000) (Cell Signaling Technology), anti-phospho-p53(serine 37) (1:1,000) (Cell Signaling Technology), anti-phospho-p53(serine 46) (1:1,000) (Cell Signaling Technology), anti-phospho-p53(serine 392) (1:1,000) (Cell Signaling Technology), anti-actin (1:4,000) (Chemicon, Temecula, CA), anti-phospho-ERK1/2 (1:1,000) (Cell Signaling Technology), anti-ERK1/2 (1:1,000) (Santa Cruz Biotechnology), anti-p53 (Pab 240 at 1:1,000) (Santa Cruz Biotechnology), anti-mitogen-activated protein kinase (MAPK) phosphatase 1 (MKP1) (C-19 at 1:500) (Santa Cruz Biotechnology), monoclonal rat anti-DJ-1 (1:1,000), polyclonal anti-DJ-1 (1:4,000), monoclonal mouse anti-DJ-1 (3E8 at 1: 4,000) (MBL), and anti-oxidized DJ-1 (1:1,000).

Techniques: Luciferase, Transfection, Expressing, Quantitative RT-PCR

Journal: Molecular and Cellular Biology

Article Title: Oxidized DJ-1 Inhibits p53 by Sequestering p53 from Promoters in a DNA-Binding Affinity-Dependent Manner

doi: 10.1128/MCB.01350-12

Figure Lengend Snippet: Phosphorylation levels of ERK in cells under conditions of oxidative stress. DJ-1+/+ and DJ-1−/− mouse cells were starved for 6 h and then treated with or without two pulses of H2O2. Proteins were analyzed by Western blotting (C), and the relative phosphorylation levels of ERK were quantified (A). DJ-1+/+ and DJ-1−/− mouse cells were transfected with control siRNA, DUSP1 siRNA-1, or DUSP1 siRNA-2, starved for 6 h at 48 h after transfection, and then treated with two pulses of H2O2. Proteins were analyzed by Western blotting (B), and the relative phosphorylation levels of ERK were quantified (D). Values are means ± SE (n = 3 experiments). *, P < 0.05.

Article Snippet: The antibodies used in this study were anti-HA (1:2,000) (MBL, Nagoya, Japan), anti-FLAG F7425 (1:1,000) (Sigma), anti-T7 (1:1,000) (Novagen, Madison, WI), anti-p53 (DO-1 at 1:1,000) (Santa Cruz Biotechnology, Santa Cruz, CA), anti-phospho-p53(serine 6) (1:1,000) (Cell Signaling Technology, Danvers, MA), anti-phospho-p53(serine 9) (1:1,000) (Cell Signaling Technology), anti-phospho-p53(serine 15) (1:1,000) (Cell Signaling Technology), anti-phospho-p53(serine 20) (1:1,000) (Cell Signaling Technology), anti-phospho-p53(serine 37) (1:1,000) (Cell Signaling Technology), anti-phospho-p53(serine 46) (1:1,000) (Cell Signaling Technology), anti-phospho-p53(serine 392) (1:1,000) (Cell Signaling Technology), anti-actin (1:4,000) (Chemicon, Temecula, CA), anti-phospho-ERK1/2 (1:1,000) (Cell Signaling Technology), anti-ERK1/2 (1:1,000) (Santa Cruz Biotechnology), anti-p53 (Pab 240 at 1:1,000) (Santa Cruz Biotechnology), anti-mitogen-activated protein kinase (MAPK) phosphatase 1 (MKP1) (C-19 at 1:500) (Santa Cruz Biotechnology), monoclonal rat anti-DJ-1 (1:1,000), polyclonal anti-DJ-1 (1:4,000), monoclonal mouse anti-DJ-1 (3E8 at 1: 4,000) (MBL), and anti-oxidized DJ-1 (1:1,000).

Techniques: Western Blot, Transfection

Journal: Molecular and Cellular Biology

Article Title: Oxidized DJ-1 Inhibits p53 by Sequestering p53 from Promoters in a DNA-Binding Affinity-Dependent Manner

doi: 10.1128/MCB.01350-12

Figure Lengend Snippet: DJ-1 regulates cell survival under conditions of oxidative stress. (A) DJ-1+/+ and DJ-1−/− mouse cells were transfected with control siRNA or DUSP1 siRNA-1 and treated as described for Fig. 11. Forty hours after the addition of H2O2, cell cycle profiles were examined by using flow cytometry. (B) The sub-G1 phase of the cell cycle shown in panel A was quantified, and the relative number of cells in the sub-G1 fraction compared to that in DJ-1+/+ cells with control siRNA and H2O2 was calculated. Values are means ± SE (n = 3 experiments). ***, P < 0.001.

Article Snippet: The antibodies used in this study were anti-HA (1:2,000) (MBL, Nagoya, Japan), anti-FLAG F7425 (1:1,000) (Sigma), anti-T7 (1:1,000) (Novagen, Madison, WI), anti-p53 (DO-1 at 1:1,000) (Santa Cruz Biotechnology, Santa Cruz, CA), anti-phospho-p53(serine 6) (1:1,000) (Cell Signaling Technology, Danvers, MA), anti-phospho-p53(serine 9) (1:1,000) (Cell Signaling Technology), anti-phospho-p53(serine 15) (1:1,000) (Cell Signaling Technology), anti-phospho-p53(serine 20) (1:1,000) (Cell Signaling Technology), anti-phospho-p53(serine 37) (1:1,000) (Cell Signaling Technology), anti-phospho-p53(serine 46) (1:1,000) (Cell Signaling Technology), anti-phospho-p53(serine 392) (1:1,000) (Cell Signaling Technology), anti-actin (1:4,000) (Chemicon, Temecula, CA), anti-phospho-ERK1/2 (1:1,000) (Cell Signaling Technology), anti-ERK1/2 (1:1,000) (Santa Cruz Biotechnology), anti-p53 (Pab 240 at 1:1,000) (Santa Cruz Biotechnology), anti-mitogen-activated protein kinase (MAPK) phosphatase 1 (MKP1) (C-19 at 1:500) (Santa Cruz Biotechnology), monoclonal rat anti-DJ-1 (1:1,000), polyclonal anti-DJ-1 (1:4,000), monoclonal mouse anti-DJ-1 (3E8 at 1: 4,000) (MBL), and anti-oxidized DJ-1 (1:1,000).

Techniques: Transfection, Flow Cytometry

Journal: Molecular and Cellular Biology

Article Title: Oxidized DJ-1 Inhibits p53 by Sequestering p53 from Promoters in a DNA-Binding Affinity-Dependent Manner

doi: 10.1128/MCB.01350-12

Figure Lengend Snippet: Schematic models of the role of DJ-1 in the inhibition of p53 transactivation. (A) DJ-1 activates cell survival pathways under conditions of oxidative stress through downregulation of DUSP1 expression. (B) DJ-1 suppresses transactivation activity of p53 depending on the p53 DNA-binding affinity.

Article Snippet: The antibodies used in this study were anti-HA (1:2,000) (MBL, Nagoya, Japan), anti-FLAG F7425 (1:1,000) (Sigma), anti-T7 (1:1,000) (Novagen, Madison, WI), anti-p53 (DO-1 at 1:1,000) (Santa Cruz Biotechnology, Santa Cruz, CA), anti-phospho-p53(serine 6) (1:1,000) (Cell Signaling Technology, Danvers, MA), anti-phospho-p53(serine 9) (1:1,000) (Cell Signaling Technology), anti-phospho-p53(serine 15) (1:1,000) (Cell Signaling Technology), anti-phospho-p53(serine 20) (1:1,000) (Cell Signaling Technology), anti-phospho-p53(serine 37) (1:1,000) (Cell Signaling Technology), anti-phospho-p53(serine 46) (1:1,000) (Cell Signaling Technology), anti-phospho-p53(serine 392) (1:1,000) (Cell Signaling Technology), anti-actin (1:4,000) (Chemicon, Temecula, CA), anti-phospho-ERK1/2 (1:1,000) (Cell Signaling Technology), anti-ERK1/2 (1:1,000) (Santa Cruz Biotechnology), anti-p53 (Pab 240 at 1:1,000) (Santa Cruz Biotechnology), anti-mitogen-activated protein kinase (MAPK) phosphatase 1 (MKP1) (C-19 at 1:500) (Santa Cruz Biotechnology), monoclonal rat anti-DJ-1 (1:1,000), polyclonal anti-DJ-1 (1:4,000), monoclonal mouse anti-DJ-1 (3E8 at 1: 4,000) (MBL), and anti-oxidized DJ-1 (1:1,000).

Techniques: Inhibition, Expressing, Activity Assay, Binding Assay