Journal: Frontiers in Cellular Neuroscience

Article Title: Obesity Increases Mitogen-Activated Protein Kinase Phosphatase-3 Levels in the Hypothalamus of Mice

doi: 10.3389/fncel.2017.00313

Figure Lengend Snippet: Increased hypothalamic Mitogen-activated Protein Kinase Phosphatase 3 (MKP-3) content in obese mice. MKP-3 protein content in the hypothalamus of lean and obese mice. The bars in the graphs represent the mean and SEM ( n = 5). ** p < 0.01 vs. Lean group.

Article Snippet: The primary antibodies used were anti-Akt (rabbit, sc-8312, Santa Cruz Biotechnology ® ), anti-phospho-Akt (Ser473; rabbit, sc-101629, Santa Cruz Biotechnology ® ), anti-FoxO1 (rabbit, 9454s, Cell Signaling Technology ® ), anti- β-Actin (rabbit, 8457, Cell Signaling Technology ® ), Anti-phospho-Erk1/Erk2 (T202/Y204/T185/Y187; rabbit, AF1018, R&D Systems ® ), anti-Erk1/2 (rabbit, AF1576, R&D Systems ® ), anti-phospho-FoxO1 (Ser256; rabbit, 9461s, Cell Signaling Technology ® ), anti-MKP-3 (rabbit, bs-11546R, Bioss ® ).

Techniques:

Journal: Frontiers in Cellular Neuroscience

Article Title: Obesity Increases Mitogen-Activated Protein Kinase Phosphatase-3 Levels in the Hypothalamus of Mice

doi: 10.3389/fncel.2017.00313

Figure Lengend Snippet: Phosphorylation of the proteins involved with the MKP-3 phosphatase activity (pFoxO1 and pErk1/2) after insulin stimulus. (A) FoxO1 phosphorylation (S256) normalized by its total content. (B) Erk1/2 phosphorylation (T202/Y204/T185/Y187) normalized by its total content. (C) Akt phosphorylation (S473) normalized by its total content. Related to the total content of FoxO1, Erk and Akt were used as a control, only Erk1/2 showed a significant increase compared to the CTL group. The bars in the graphs represent the mean and SEM ( n = 6). * p < 0.05 vs. Lean group. *** p < 0.001 vs. Lean group.

Article Snippet: The primary antibodies used were anti-Akt (rabbit, sc-8312, Santa Cruz Biotechnology ® ), anti-phospho-Akt (Ser473; rabbit, sc-101629, Santa Cruz Biotechnology ® ), anti-FoxO1 (rabbit, 9454s, Cell Signaling Technology ® ), anti- β-Actin (rabbit, 8457, Cell Signaling Technology ® ), Anti-phospho-Erk1/Erk2 (T202/Y204/T185/Y187; rabbit, AF1018, R&D Systems ® ), anti-Erk1/2 (rabbit, AF1576, R&D Systems ® ), anti-phospho-FoxO1 (Ser256; rabbit, 9461s, Cell Signaling Technology ® ), anti-MKP-3 (rabbit, bs-11546R, Bioss ® ).

Techniques: Activity Assay

Journal: Frontiers in Cellular Neuroscience

Article Title: Obesity Increases Mitogen-Activated Protein Kinase Phosphatase-3 Levels in the Hypothalamus of Mice

doi: 10.3389/fncel.2017.00313

Figure Lengend Snippet: Transcriptome and phenotype bioinformatics analysis. (A) Positive Pearson’s correlation between hypothalamic MKP-3 mRNA in lean mice and body weight ( n = 11). (B) Negative Pearson’s correlation between hypothalamic MKP-3 mRNA and oxygen consumption (VO 2 ) in light phase ( n = 19).

Article Snippet: The primary antibodies used were anti-Akt (rabbit, sc-8312, Santa Cruz Biotechnology ® ), anti-phospho-Akt (Ser473; rabbit, sc-101629, Santa Cruz Biotechnology ® ), anti-FoxO1 (rabbit, 9454s, Cell Signaling Technology ® ), anti- β-Actin (rabbit, 8457, Cell Signaling Technology ® ), Anti-phospho-Erk1/Erk2 (T202/Y204/T185/Y187; rabbit, AF1018, R&D Systems ® ), anti-Erk1/2 (rabbit, AF1576, R&D Systems ® ), anti-phospho-FoxO1 (Ser256; rabbit, 9461s, Cell Signaling Technology ® ), anti-MKP-3 (rabbit, bs-11546R, Bioss ® ).

Techniques:

Journal: Frontiers in Cellular Neuroscience

Article Title: Obesity Increases Mitogen-Activated Protein Kinase Phosphatase-3 Levels in the Hypothalamus of Mice

doi: 10.3389/fncel.2017.00313

Figure Lengend Snippet: Schematic model summarizing the possible molecular mechanisms by which MKP-3 led to food intake increase and thermogenesis decrease. The obese mice presented high content of MKP-3 in the hypothalamus, and decreased phosphorylation of FoxO1 and Erk1/2 leading to decreased oxygen consumption, heat production, spontaneous activity, RER and increased food intake.

Article Snippet: The primary antibodies used were anti-Akt (rabbit, sc-8312, Santa Cruz Biotechnology ® ), anti-phospho-Akt (Ser473; rabbit, sc-101629, Santa Cruz Biotechnology ® ), anti-FoxO1 (rabbit, 9454s, Cell Signaling Technology ® ), anti- β-Actin (rabbit, 8457, Cell Signaling Technology ® ), Anti-phospho-Erk1/Erk2 (T202/Y204/T185/Y187; rabbit, AF1018, R&D Systems ® ), anti-Erk1/2 (rabbit, AF1576, R&D Systems ® ), anti-phospho-FoxO1 (Ser256; rabbit, 9461s, Cell Signaling Technology ® ), anti-MKP-3 (rabbit, bs-11546R, Bioss ® ).

Techniques: Activity Assay

Journal:

Article Title: Inhibition of Mitogen-activated Protein Kinase Phosphatase 3 Activity by Interdomain Binding *

doi: 10.1074/jbc.M801747200

Figure Lengend Snippet: Detection of expressed recombinant proteins. MKP3 protein expression was detected by Western analysis and SDS-PAGE. A, Western analysis. MKP3 variants were separated on 16% SDS-PAGE and then transferred to Immobilon-P membrane for immunodetection using an anti-His6 antibody. Lane M, His6-tagged molecular size markers; lane 1, MKP3:CT (31 kDa); lane 2, MKP3:NT (18 kDa); lane 3, MKP3:WT (45 kDa). B, SDS-PAGE separation. Following protein purification, the MKP3 variants were separated by 12% SDS-PAGE. Lane M, Molecular size markers; lane 1, MKP3:WT; lane 2, MKP3:NT; lane 3, MKP3:CT; lane M, molecular size markers.

Article Snippet: Blots were probed with anti-human MKP3 mouse monoclonal antibody (1 μg/ml; catalogue No. MAB3576, R&D Systems, Minneapolis, MN), rabbit polyclonal anti-human p44/42 antibody 9102 (1:5000; Cell Signaling Technology, Danvers, MA), rabbit monoclonal anti-human phosphorylated p44/42 Thr-202/Tyr-204 antibody 197G2 (1:1000; Cell Signaling Technology), and revealed with horseradish peroxidase-conjugated secondary antibodies (1:2000; Amersham Biosciences) using 1:4 diluted SuperSignal reagent (Pierce).

Techniques: Recombinant, Expressing, Western Blot, SDS Page, Membrane, Immunodetection, Protein Purification

Journal:

Article Title: Inhibition of Mitogen-activated Protein Kinase Phosphatase 3 Activity by Interdomain Binding *

doi: 10.1074/jbc.M801747200

Figure Lengend Snippet: Secondary structure analysis by circular dichroism spectroscopy. Folding of the recombinant MKP3 proteins was assessed using circular dichroism spectroscopy to determine the secondary structure composition. Data shows the residue-weighted CD spectra traces from MKP3:NT, MKP3:CT, and MKP3:WT. The spectra were deconvoluted using DICHROWEB as described under “Experimental Procedures” to obtain the average secondary structure content from each recombinant protein.

Article Snippet: Blots were probed with anti-human MKP3 mouse monoclonal antibody (1 μg/ml; catalogue No. MAB3576, R&D Systems, Minneapolis, MN), rabbit polyclonal anti-human p44/42 antibody 9102 (1:5000; Cell Signaling Technology, Danvers, MA), rabbit monoclonal anti-human phosphorylated p44/42 Thr-202/Tyr-204 antibody 197G2 (1:1000; Cell Signaling Technology), and revealed with horseradish peroxidase-conjugated secondary antibodies (1:2000; Amersham Biosciences) using 1:4 diluted SuperSignal reagent (Pierce).

Techniques: Circular Dichroism, Spectroscopy, Recombinant, Residue

Journal:

Article Title: Inhibition of Mitogen-activated Protein Kinase Phosphatase 3 Activity by Interdomain Binding *

doi: 10.1074/jbc.M801747200

Figure Lengend Snippet: Summary of surface plasmon resonance data Surface plasmon resonance experiments were performed on MKP3 (WT, NT, and CT) and MAPK (ERK2 and JNK) proteins that were covalently bound to SPR sensor chips. Binding data was obtained with increasing concentrations of either MKP3:WT or MKP3:CT. Binding curves were fitted to a 1:1 Langmuir binding model [ A ] + [ B ] ↔ [ AB ] using the BIAevaluation 4.0 program to obtain association and dissociation constants.

Article Snippet: Blots were probed with anti-human MKP3 mouse monoclonal antibody (1 μg/ml; catalogue No. MAB3576, R&D Systems, Minneapolis, MN), rabbit polyclonal anti-human p44/42 antibody 9102 (1:5000; Cell Signaling Technology, Danvers, MA), rabbit monoclonal anti-human phosphorylated p44/42 Thr-202/Tyr-204 antibody 197G2 (1:1000; Cell Signaling Technology), and revealed with horseradish peroxidase-conjugated secondary antibodies (1:2000; Amersham Biosciences) using 1:4 diluted SuperSignal reagent (Pierce).

Techniques: SPR Assay, Binding Assay

Journal:

Article Title: Inhibition of Mitogen-activated Protein Kinase Phosphatase 3 Activity by Interdomain Binding *

doi: 10.1074/jbc.M801747200

Figure Lengend Snippet: Biacore analysis of association kinetics. Binding between the N- and C-terminal domains of MKP3 was measured using surface plasmon resonance. MKP3:WT, MKP3:NT, and MKP3:CT were immobilized on BIAcore SPR sensor chips with MKP3:CT used in the mobile phase. A, MKP3:WT; B, MKP3: NT; C, MKP3:CT. Different curves for each figure reflect increasing MKP3:CT concentrations (0-25 μm). These data indicate that increasing concentrations of MKP3:CT lead to an increase in the SPR plateau phase response expressed as resonance units (RU). These data were analyzed by curve fitting to a 1:1 Langmuir association to determine ka and KD values (Table 1).

Article Snippet: Blots were probed with anti-human MKP3 mouse monoclonal antibody (1 μg/ml; catalogue No. MAB3576, R&D Systems, Minneapolis, MN), rabbit polyclonal anti-human p44/42 antibody 9102 (1:5000; Cell Signaling Technology, Danvers, MA), rabbit monoclonal anti-human phosphorylated p44/42 Thr-202/Tyr-204 antibody 197G2 (1:1000; Cell Signaling Technology), and revealed with horseradish peroxidase-conjugated secondary antibodies (1:2000; Amersham Biosciences) using 1:4 diluted SuperSignal reagent (Pierce).

Techniques: Binding Assay, SPR Assay

Journal:

Article Title: Inhibition of Mitogen-activated Protein Kinase Phosphatase 3 Activity by Interdomain Binding *

doi: 10.1074/jbc.M801747200

Figure Lengend Snippet: Recombinant MKP3 steady state analysis MKP3 proteins were assayed using p NPP as a colorimetric substrate in the presence or absence of ERK2 and DMSO, which were used to activate the MKP3 phosphatase function. MKP3 proteins were tested using 0-50 m m substrate (for nonactive MKP3 tests) or 0-10 m m substrate (for ERK- or DMSO-activated tests). To detect MKP3 phosphatase activity, the hydrolysis of substrate was measured at 405 nm following a 60-min incubation at 25 °C. To determine k cat and K m , the data were analyzed using GraphPad Prizm 4.0.

Article Snippet: Blots were probed with anti-human MKP3 mouse monoclonal antibody (1 μg/ml; catalogue No. MAB3576, R&D Systems, Minneapolis, MN), rabbit polyclonal anti-human p44/42 antibody 9102 (1:5000; Cell Signaling Technology, Danvers, MA), rabbit monoclonal anti-human phosphorylated p44/42 Thr-202/Tyr-204 antibody 197G2 (1:1000; Cell Signaling Technology), and revealed with horseradish peroxidase-conjugated secondary antibodies (1:2000; Amersham Biosciences) using 1:4 diluted SuperSignal reagent (Pierce).

Techniques: Recombinant, Activity Assay, Incubation

Journal:

Article Title: Inhibition of Mitogen-activated Protein Kinase Phosphatase 3 Activity by Interdomain Binding *

doi: 10.1074/jbc.M801747200

Figure Lengend Snippet: DMSO concentration dependence of MKP3 catalytic domain. As a first step in the development of an ERK-free MKP3:CT activity assay, the ability of MKP3:CT and MKP3:WT to hydrolyze pNPP was tested in the presence of increasing DMSO concentration. DMSO, which has been shown to increase the activity of full-length MKP3, was observed to increase the phosphatase activity of both MKP3:CT and MKP3:WT, showing that DMSO can be used as a chemical activator to the binding-deficient MKP3:CT mutant in the absence of ERK.

Article Snippet: Blots were probed with anti-human MKP3 mouse monoclonal antibody (1 μg/ml; catalogue No. MAB3576, R&D Systems, Minneapolis, MN), rabbit polyclonal anti-human p44/42 antibody 9102 (1:5000; Cell Signaling Technology, Danvers, MA), rabbit monoclonal anti-human phosphorylated p44/42 Thr-202/Tyr-204 antibody 197G2 (1:1000; Cell Signaling Technology), and revealed with horseradish peroxidase-conjugated secondary antibodies (1:2000; Amersham Biosciences) using 1:4 diluted SuperSignal reagent (Pierce).

Techniques: Concentration Assay, Activity Assay, Binding Assay, Mutagenesis

Journal:

Article Title: Inhibition of Mitogen-activated Protein Kinase Phosphatase 3 Activity by Interdomain Binding *

doi: 10.1074/jbc.M801747200

Figure Lengend Snippet: Inhibition of enzyme activity by N-terminal MKP3. The effect of MKP3:NT binding on activated MKP3:CT was tested using the DMSO-based phosphatase assay with increasing concentrations of MKP3:NT. Nonactive MKP3:CT (○, MKP3:CT/H2O) was used as a base line for MKP3:CT activity. DMSO-activated MKP3:CT (▵, MKP3:CT/DMSO) was tested in the absence of MKP3:NT and set to 100% activity. The MKP3:NT concentration was then increased to a 2.2:1 concentration relative to MKP3:CT (plotted on the logarithmic scale). The increased MKP3:NT led to a dose-dependant decrease in MKP3:CT activity. The same experiment was performed on DMSO-activated MKP3:WT (□, MKP3:WT/DMSO), which showed that increasing MKP3:NT was capable of further inhibiting the full-length MKP3 activity.

Article Snippet: Blots were probed with anti-human MKP3 mouse monoclonal antibody (1 μg/ml; catalogue No. MAB3576, R&D Systems, Minneapolis, MN), rabbit polyclonal anti-human p44/42 antibody 9102 (1:5000; Cell Signaling Technology, Danvers, MA), rabbit monoclonal anti-human phosphorylated p44/42 Thr-202/Tyr-204 antibody 197G2 (1:1000; Cell Signaling Technology), and revealed with horseradish peroxidase-conjugated secondary antibodies (1:2000; Amersham Biosciences) using 1:4 diluted SuperSignal reagent (Pierce).

Techniques: Inhibition, Activity Assay, Binding Assay, Phosphatase Assay, Concentration Assay

Journal:

Article Title: Inhibition of Mitogen-activated Protein Kinase Phosphatase 3 Activity by Interdomain Binding *

doi: 10.1074/jbc.M801747200

Figure Lengend Snippet: Analysis of MKP3 and ERK1/2 expression in human pancreatic andenocarcinoma cell lines. MKP3 and ERK1/2 proteins were detected using Western blot hybridization and laser densitometry as described under “Experimental Procedures” to confirm their presence in pancreatic adenocarcinomas. A, Western blot was performed on 20 μg of total cellular protein from each of the indicated cell lines. RAS-associated nuclear protein (RAN) was used as the protein loading control. Lane C, HeLa cells (positive control); lane 1, normal pancreas (homogenized human pancreatic tissue used to detect the base-line MKP3 level); lane 2, HPDE4 (immortalized, nontumorigenic ductal pancreatic cells); lane 3, HPDE6 (immortalized, nontumorigenic ductal pancreatic cells); lanes 4-11, utilized pancreatic adenocarcinoma cell lines. Lane 4, CRL1420 (MIAPaCa-2); lane 5, CRL1469; lane 6, CRL1682 (BxPC-3); lane 7, CRL1687; lane 8, CRL1837 (Su86.86); lane 9, HTB79; lane 10, HTB80; lane 11, HTB134; lane 12, HTB147. Immunodetection for each specified protein is shown. Densitometric data, adjusted to the RAS-associated nuclear protein loading control, are shown relative to HPDE6, which was set to 1. B, Mkp3 Northern blot analysis. Total cellular RNA was fractionated and processed as described under “Experimental Procedures.” Mkp3 detection was confirmed using 1.25 kbp of human Mkp3 HindIII/XhoI cDNA fragment, and human β-actin cDNA was used for the loading control. Lane 1, normal human pancreas; lane 2, CRL1420 (MIAPaCa-2); lane 3, CRL 1469; lane 4, 1682; lane 5, 1687 (BxPC-3); lane 6, 1837 (Su86.86); lane 7, HTB79; lane 8, HTB80; lane 9, HTB134; lane 10, HTB147. The Northern blot correlated well with the Western blot, indicating that Mkp3 RNA was actively transcribed in seven of the nine pancreatic adenocarcinomas tested.

Article Snippet: Blots were probed with anti-human MKP3 mouse monoclonal antibody (1 μg/ml; catalogue No. MAB3576, R&D Systems, Minneapolis, MN), rabbit polyclonal anti-human p44/42 antibody 9102 (1:5000; Cell Signaling Technology, Danvers, MA), rabbit monoclonal anti-human phosphorylated p44/42 Thr-202/Tyr-204 antibody 197G2 (1:1000; Cell Signaling Technology), and revealed with horseradish peroxidase-conjugated secondary antibodies (1:2000; Amersham Biosciences) using 1:4 diluted SuperSignal reagent (Pierce).

Techniques: Expressing, Western Blot, Hybridization, Control, Positive Control, Immunodetection, Northern Blot

Journal:

Article Title: Inhibition of Mitogen-activated Protein Kinase Phosphatase 3 Activity by Interdomain Binding *

doi: 10.1074/jbc.M801747200

Figure Lengend Snippet: Western analysis of MKP3 under nondenaturing condition. Western analysis was performed to test for MKP3 oligomerization in pancreatic adenocarcinoma cell lines and purified recombinant MKP3. Proteins were prepared in nondenaturing radioimmune precipitation assay buffer to retain any bound complexes. Cell lysate (10 μg) or purified recombinant MKP3 (0.4 μg) was separated by 7.5% SDS-PAGE prior to Western detection. A, Sypro-stained 7.5% SDS-PAGE separation of MKP3:WT protein standards. Lane 1, molecular size markers; lane 2, purified monomeric MKP3:WT; lane 3, purified MKP3:WT allowed to oligomerize. B, Western-detected MKP3 proteins. Lane 1, CRL1687; lane 2, purified MKP3:WT; lane 3, purified MKP3:CT. C, Western-detected MKP3 proteins in pancreatic adenocarcinoma cell lysates. Lane 1, HPDE6; lane 2, CRL1420; lane 3, CRL1469; lane 4, CRL1682; lane 5, CRL1687; lane 6, CRL1837; lane 7, HTB80; lane 8, HTB134; lane 9, HTB147.

Article Snippet: Blots were probed with anti-human MKP3 mouse monoclonal antibody (1 μg/ml; catalogue No. MAB3576, R&D Systems, Minneapolis, MN), rabbit polyclonal anti-human p44/42 antibody 9102 (1:5000; Cell Signaling Technology, Danvers, MA), rabbit monoclonal anti-human phosphorylated p44/42 Thr-202/Tyr-204 antibody 197G2 (1:1000; Cell Signaling Technology), and revealed with horseradish peroxidase-conjugated secondary antibodies (1:2000; Amersham Biosciences) using 1:4 diluted SuperSignal reagent (Pierce).

Techniques: Western Blot, Purification, Recombinant, SDS Page, Staining

Journal:

Article Title: Inhibition of Mitogen-activated Protein Kinase Phosphatase 3 Activity by Interdomain Binding *

doi: 10.1074/jbc.M801747200

Figure Lengend Snippet: Relative amounts of monomer, dimer, trimer, and oligomer in pancreatic adenocarcinoma cells Western blot analysis was performed on pancreatic adenocarcinoma cell lysates as described under “Experimental Procedures.” The total MKP3 and MKP3 band pixel counts were determined by densitometry, and then the relative count for each band was used to obtain a percentage of MKP3 existing as monomer, dimer, trimer, and oligomer.

Article Snippet: Blots were probed with anti-human MKP3 mouse monoclonal antibody (1 μg/ml; catalogue No. MAB3576, R&D Systems, Minneapolis, MN), rabbit polyclonal anti-human p44/42 antibody 9102 (1:5000; Cell Signaling Technology, Danvers, MA), rabbit monoclonal anti-human phosphorylated p44/42 Thr-202/Tyr-204 antibody 197G2 (1:1000; Cell Signaling Technology), and revealed with horseradish peroxidase-conjugated secondary antibodies (1:2000; Amersham Biosciences) using 1:4 diluted SuperSignal reagent (Pierce).

Techniques: Western Blot

Journal:

Article Title: Inhibition of Mitogen-activated Protein Kinase Phosphatase 3 Activity by Interdomain Binding *

doi: 10.1074/jbc.M801747200

Figure Lengend Snippet: In vitro kinetic and post-transfection biological activity of recombinant MKP3/V5 enzyme. A, V5 epitope-tagged MKP3 was purified from E. coli and subjected to steady state kinetic analysis under DMSO(□) or ERK2 (▵) inducing conditions or under noninducing condition (○) as described under “Experimental Procedures.” Both DMSO and ERK2 were capable of increasing the catalytic efficiency of MKP3, resulting in an increased vmax and decreased Km relative to the uninduced control. B, effect of transient MKP3/V5 up-regulation on ERK1/2 and phosphorylated ERK1/2 levels in human pancreatic adenocarcinoma cell lines. CRL1469 and CRL1682 cultures were co-transfected with 2.5 μg of each form I pVgRXR and pInd/GS/MK3/V5 as described under “Experimental Procedures.” V5 epitope-tagged MKP3 expression was induced at 16 h post-transfection with 5 μm pronasterone A. Cell lysates were prepared 48 h later and processed for Western blot hybridization and laser densitometry as described under “Experimental Procedures.” Epitope-tagged MKP3 was detected with a mouse monoclonal anti-V5 antibody. Dramatic up-regulation of catalytically active epitope-tagged MKP3 had no significant effect on ERK1/2 or phosphorylated ERK1/2 levels. C, cell morphology and general culture health of MKP3/V5-transfected and pronasterone A-induced CRL1469 and CRL1682 adenocarcinoma cells. Transfected cells were photographed under phase contrast 48 h following pronasterone A induction. Cell morphology and viability were unaffected despite up-regulation of epitope-tagged MKP3.

Article Snippet: Blots were probed with anti-human MKP3 mouse monoclonal antibody (1 μg/ml; catalogue No. MAB3576, R&D Systems, Minneapolis, MN), rabbit polyclonal anti-human p44/42 antibody 9102 (1:5000; Cell Signaling Technology, Danvers, MA), rabbit monoclonal anti-human phosphorylated p44/42 Thr-202/Tyr-204 antibody 197G2 (1:1000; Cell Signaling Technology), and revealed with horseradish peroxidase-conjugated secondary antibodies (1:2000; Amersham Biosciences) using 1:4 diluted SuperSignal reagent (Pierce).

Techniques: In Vitro, Transfection, Activity Assay, Recombinant, Purification, Control, Expressing, Western Blot, Hybridization

Journal:

Article Title: Inhibition of Mitogen-activated Protein Kinase Phosphatase 3 Activity by Interdomain Binding *

doi: 10.1074/jbc.M801747200

Figure Lengend Snippet: Models of ERK-induced activation and function in MKP3. The results of the binding and autoinhibition studies support a model of MKP3 deactivation due to oligomerization of MKP3. A, under normal conditions, the presence of ERK (purple) leads to competitive binding and a two-step MKP3 activation: displacement of the C-terminal domain (yellow oval) from the N-terminal domain (green oval) followed by allosteric activation of the C-terminal domain (yellow square) by the N-terminally bound ERK. The allosteric activation results in dephosphorylation of phosphotyrosine and phosphothreonine residues on ERK (red circles). B, if MKP3 is overexpressed, it undergoes interdomain binding, leading to the formation of high molecular mass oligomers. C, ERK binding to the N-terminal domain of the oligomerized MKP3 can release C-terminal catalytic domain, but this catalytic domain may be unable to become allosterically activated or to bind and, consequently, dephosphorylate ERK. Thus, the presence of oligomers adversely affects the ability of MKP3 to dephosphorylate ERK.

Article Snippet: Blots were probed with anti-human MKP3 mouse monoclonal antibody (1 μg/ml; catalogue No. MAB3576, R&D Systems, Minneapolis, MN), rabbit polyclonal anti-human p44/42 antibody 9102 (1:5000; Cell Signaling Technology, Danvers, MA), rabbit monoclonal anti-human phosphorylated p44/42 Thr-202/Tyr-204 antibody 197G2 (1:1000; Cell Signaling Technology), and revealed with horseradish peroxidase-conjugated secondary antibodies (1:2000; Amersham Biosciences) using 1:4 diluted SuperSignal reagent (Pierce).

Techniques: Activation Assay, Binding Assay, De-Phosphorylation Assay

Journal: PLoS ONE

Article Title: Differential Roles for DUSP Family Members in Epithelial-to-Mesenchymal Transition and Cancer Stem Cell Regulation in Breast Cancer

doi: 10.1371/journal.pone.0148065

Figure Lengend Snippet: DUSP1 , DUSP4 , and DUSP6 transcript levels measured by real-time PCR in ( A ) MCF-7 and MDA-MB-231 cells. Data are expressed as arbitrary copy numbers normalised to PPIA and are representative of the mean ± SE. ( B ) MCF-7 cells after incubation with PMA for various periods of time. Spline curves represent mean arbitrary copy numbers normalised to PPIA of two independent experiments. ( C ) MCF-7 cells after incubation with vehicle alone or in the presence of either PMA or PMA+TGF-β for 60 h. Data are expressed as relative fold change to MCF-7 normalised to PPIA and are representative of the mean ± SE of four independent experiments. MCF-7 cells were incubated with vehicle alone or in the presence of PMA for 60 h. Transcript levels measured by real-time PCR of: ( D ) CDH1 , VIM , FN1 , SNAI1 , SNAI2 , CD44 , and PLAUR and ( E ) DUSP1 , DUSP4 , and DUSP6 . Data are presented as relative fold change to MCF-7 normalised to PPIA and are representative of the mean ± SE. ( F ) DUSP1 , DUSP4 , and DUSP6 transcript levels in PKC-θ siRNA transfected cells. Data are expressed as relative fold change to MCF-7 MOCK siRNA normalised to PPIA and are representative of the mean ± SE for four experiments. ( G ) PKC-θ ChIP-seq across DUSP1 , DUSP4 , and DUSP6 transcripts in MCF-7 and MCF-7/PMA cells. Data are shown in the UCSC Genome Browser. The scale in all UCSC images is indicated on the y-axis as numbers in reads per million mapped reads.

Article Snippet: Forward transfection reactions were performed for 6 h with 50 nM human DUSP1 siRNA (sc-35937), DUSP4 siRNA (sc-38998), DUSP6 siRNA (sc-39000), 20 nM PKC-θ siRNA (sc-36252), and 10 nM MOCK siRNA (sc-36869) (Santa Cruz) using Lipofectamine 2000 (Invitrogen).

Techniques: Real-time Polymerase Chain Reaction, Incubation, Transfection, ChIP-sequencing

Journal: PLoS ONE

Article Title: Differential Roles for DUSP Family Members in Epithelial-to-Mesenchymal Transition and Cancer Stem Cell Regulation in Breast Cancer

doi: 10.1371/journal.pone.0148065

Figure Lengend Snippet: Confocal laser scanning microscopy was performed on either MDA-MB-231 cells or MCF-7 cells treated with vehicle alone or PMA for 60 h. Cells were fixed and probed with primary rabbit, mouse, or goat antibodies to DUSP1, DUSP4, and DUSP6, respectively, followed by the corresponding secondary antibody conjugated to Alexa-Fluor 568 or Alexa-Fluor 488. Fn/c values for: ( A ) MCF-7 and MCF-7/PMA cells; ( B ) MDA-MB-231 cells. Data shown represent the mean ± SE. Value 1 = equal cytoplasmic and nuclear fractions; >1 = nuclear bias; <1 = cytoplasmic bias. MCF-7 cells were probed with either: ( C ) primary anti-rabbit-DUSP1 and anti-mouse-H3K9me1; or ( D ) primary anti-mouse-DUSP4 and anti-rabbit-H3K27ac or anti-rabbit-H3K4me1. All antibodies were conjugated to Alexa-Fluor 568 or Alexa-Fluor 488. MDA-MB-231 cells probed with either: ( E ) primary anti-rabbit-DUSP1 and anti-mouse-H3K4me3 or anti-mouse-H3K9me1 or ( F ) primary anti-mouse-DUSP4 and anti-rabbit-H3K4me1 or anti-rabbit-H3K27ac. All antibodies were conjugated to Alexa-Fluor 568 or Alexa-Fluor 488. Representative images for each dataset are shown: green = DUSPs; red = histone marks; yellow = overlap between DUSPs and histone mark fluorescence signals. The plot-profile feature of ImageJ was used to plot the fluorescence signal intensity along a single line spanning the nucleus (n = 5 lines per nucleus, 5 individual cells). The average fluorescence signal intensity for the indicated pair of antibodies was plotted for each point on the line ±SE. Signal was plotted to compare how the signals for each antibody varied compared to the opposite antibody. The PCC was determined for each plot profile. PCC indicates the strength of relation between the two fluorochrome signals for at least 20 individual cells ± SE. Colours from representative images correspond to plot profiles.

Article Snippet: Forward transfection reactions were performed for 6 h with 50 nM human DUSP1 siRNA (sc-35937), DUSP4 siRNA (sc-38998), DUSP6 siRNA (sc-39000), 20 nM PKC-θ siRNA (sc-36252), and 10 nM MOCK siRNA (sc-36869) (Santa Cruz) using Lipofectamine 2000 (Invitrogen).

Techniques: Confocal Laser Scanning Microscopy, Fluorescence

Journal: PLoS ONE

Article Title: Differential Roles for DUSP Family Members in Epithelial-to-Mesenchymal Transition and Cancer Stem Cell Regulation in Breast Cancer

doi: 10.1371/journal.pone.0148065

Figure Lengend Snippet: MCF-7 cells were transfected with MOCK, DUSP1, DUSP4, or DUSP6 siRNAs and subsequently incubated with either vehicle or in the presence of PMA for 60 h. ( A ) Percentage of CD44 hi /CD24 lo /EpCAM + CSC-like cells as measured by flow cytometry after transfection ± SE. Numbers above each column represent percentage inhibition (-) or promotion (+) relative to MOCK siRNA for three independent experiments. MCF-7 and MDA-MB-231 were inhibited with either NSC 95397 or triptolide for 24 h. ( B ) MCF-7 cells were then incubated with either vehicle alone or PMA for 60 h. Bar graph indicates percentage inhibition of CD44 hi /CD24 lo /EpCAM + CSC-like MCF-7, MCF-7/PMA AD , and MCF-7/PMA SUS cells after inhibition ± SE for two independent experiments. ( C ) Bar graph indicates CD44 hi /CD24 lo /EpCAM + CSC-like MDA-MB-231 cells after inhibition ± SE for two independent experiments. ( D ) mRNA levels CDH1 , VIM , and FN1 in MDA-MB-231 cells after inhibition. Data represent the mean relative fold change to untreated cells normalised to PPIA ± SE for two independent experiments.

Article Snippet: Forward transfection reactions were performed for 6 h with 50 nM human DUSP1 siRNA (sc-35937), DUSP4 siRNA (sc-38998), DUSP6 siRNA (sc-39000), 20 nM PKC-θ siRNA (sc-36252), and 10 nM MOCK siRNA (sc-36869) (Santa Cruz) using Lipofectamine 2000 (Invitrogen).

Techniques: Transfection, Incubation, Flow Cytometry, Inhibition

Journal: PLoS ONE

Article Title: Differential Roles for DUSP Family Members in Epithelial-to-Mesenchymal Transition and Cancer Stem Cell Regulation in Breast Cancer

doi: 10.1371/journal.pone.0148065

Figure Lengend Snippet: DUSP6 expression in a breast cancer cell line and breast cancer tissues were examined by immunohistochemistry. ( A ) Table summarising DUSP6 expression in the indicated breast tissues. ( B ) Cell block of MDA-MB-231 cells (x630). ( C ) Grade 2 ER + /PR + /HER2 - breast cancer (x400). ( D ) Grade 3 triple-negative (ER - /PR - /HER2 - ) breast cancer (x400). ( E ) Grade 3 ER + /PR + /HER2 + breast cancer (x400) with benign breast duct. ( F ) Grade 3 ER - /PR - /HER2 + breast cancer (x400).

Article Snippet: Forward transfection reactions were performed for 6 h with 50 nM human DUSP1 siRNA (sc-35937), DUSP4 siRNA (sc-38998), DUSP6 siRNA (sc-39000), 20 nM PKC-θ siRNA (sc-36252), and 10 nM MOCK siRNA (sc-36869) (Santa Cruz) using Lipofectamine 2000 (Invitrogen).

Techniques: Expressing, Immunohistochemistry, Blocking Assay

Journal: Nature medicine

Article Title: Sex-Specific Transcriptional Signatures in Human Depression

doi: 10.1038/nm.4386

Figure Lengend Snippet: Gene co-expression modules in females with MDD are enriched for DEGs across brain regions. a, Topological overlap matrix (TOM) plots for control and b, MDD modules in females. Light color represents low topological overlap and progressively darker red color represents higher overlap. Each module is assigned by unique color. c, Circos plots displaying the degree of enrichment for DEGs (p<0.05) in female modules. Colors within squares of the plots represent the corrected FET p-value of the enrichment of DEGs across modules which are depicted in the color bar below the circos plot. Legend on the top right corner defines individual layers of the circos plot. d, Gray26 module in female MDD shows enrichment for DEGs across brain regions. Hubs and nodes are defined by the size of the circles with colors representing enrichment for DEGs across brain regions (depicted in the bottom right panel). DUSP6, labelled in red, was selected for sex-specific in vivo functional validation studies.

Article Snippet: DUSP6 and EMX1 coding regions were excised from DUSP6 (Origene cat# MR222688) and EMX1 (Origene cat# RC208006) expression clones and cloned into a p1005+ HSV vector to induce overexpression in the brain.

Techniques: Expressing, In Vivo, Functional Assay

Journal: Nature medicine

Article Title: Sex-Specific Transcriptional Signatures in Human Depression

doi: 10.1038/nm.4386

Figure Lengend Snippet: DUSP6 downregulation in vmPFC induces a sex-specific depressive-like phenotype associated with increased ERK signaling in females. a, Schematic representation of the behavioral paradigm used to assess the impact DUSP6 downregulation in males and females. b, HSV-mediated downregulation of DUSP6 covering the infra- and prelimbic regions of the vmPFC in mice. c, Behavioral consequence of DUSP6 viral downregulation in the novelty-supressed feeding test in females and d, males and in the sucrose preference test in e, females and f, males. Significance in b was assessed using independent sample t-test. Significance in tests c–f was determined using two-way ANOVA with Tuckey correction. b n=5/condition, c–d n =10/condition, e–f n=7/condition. Bars, mean ± sem; * p<0.05. g, Phospho-ERK1/2 levels assessed by Western blot in male (blue) and female (pink) mice in vmPFC with and without stress (21 days CVS). h, Total ERK1/2 protein levels assessed by Western blot in male (blue) and female (pink) mice in vmPFC with and without stress (21 days CVS). i, Representative blot of phospho-ERK1/2, total ERK1/2 and actin in male and female mice with and without stress (21 days CVS). j, Phospho-ERK1/2 levels assessed by Western blot in males (blue) and females (pink) with and without MDD in vmPFC. k, Total ERK1/2 levels assessed by Western blot in males (blue) and females (pink) vmPFC with and without MDD. l, Representative blot of phospho-ERK1/2, total ERK1/2 and actin in males and females with and without MDD. Significance in tests g–h and j–k was determined using independent sample t-test (two-tail in mice; one tail in humans). g–h n=12/stressed mice and n=10 in control conditions. j–k, Male CTRL n=18, Male MDD n=19, Female CTRL n=14, Female MDD n=18. Bars, mean ± sem; * p<0.05. m, Phospho-ERK1/2 (red) in females with MDD co-localizes in CaMKII reactive pyramidal cells (green; upper panel) but not in GAD67 reactive cells (green; lower panel). n, Phospho-ERK1/2 (purple) in female stressed mice co-localizes in CaMKII reactive pyramidal cells (red) but not in GAD67 reactive cells (green).

Article Snippet: DUSP6 and EMX1 coding regions were excised from DUSP6 (Origene cat# MR222688) and EMX1 (Origene cat# RC208006) expression clones and cloned into a p1005+ HSV vector to induce overexpression in the brain.

Techniques: Western Blot

Journal: Nature medicine

Article Title: Sex-Specific Transcriptional Signatures in Human Depression

doi: 10.1038/nm.4386

Figure Lengend Snippet: DUSP6 downregulation alters the physiological properties of vmPFC pyramidal neurons in a sex-specific fashion. a, Representative action potential traces of burst firing pyramidal neuron (upper), regular firing pyramidal neuron (middle) and interneuron (lower). b, Representative traces of neuronal activity in non-infected, GFP-infected and DUSP6 KD-infected pyramidal neurons of vmPFC of female mice. c, DUSP6 KD-induced changes of spontaneous excitatory postsynaptic current (sEPSC) frequency in Hertz (Hz) and d, amplitude in picoAmpere (pA) in female vmPFC. e, Representative traces of neuronal activity in non-infected, GFP-infected and DUSP6 KD-infected pyramidal neurons of vmPFC of male mice. f, DUSP6 KD effects on sEPSC frequency and g, amplitude in male vmPFC. Significance in tests b–c and e–f was determined using two-way ANOVA with Tuckey correction. c–d Female non-infected: n=10 mice and n=25 cells, Female GFP-infected: n=4 mice and n=17 cells, Female DUSP6 KD-infected: n=5 mice and n=24 cells. f–g, Male non-infected: n=9 mice and n=32 cells, Male GFP-infected: n=5 mice and n=29 cells, Male DUSP6 KD-infected: n=4 and n=30 cells. Bars, mean ± sem; * p<0.05.

Article Snippet: DUSP6 and EMX1 coding regions were excised from DUSP6 (Origene cat# MR222688) and EMX1 (Origene cat# RC208006) expression clones and cloned into a p1005+ HSV vector to induce overexpression in the brain.

Techniques: Activity Assay, Infection

Journal: Nature medicine

Article Title: Sex-Specific Transcriptional Signatures in Human Depression

doi: 10.1038/nm.4386

Figure Lengend Snippet: DUSP6 downregulation in female vmPFC reproduces the transcriptional and network alterations induced by 21 days of CVS in female vmPFC. a, Schematic representation of the behavioral paradigm used to assess the impact of DUSP6 downregulation in female mice. b, Transcriptional reorganization of the female-specific Gray26 gene network by DUSP6 downregulation in female vmPFC. Hubs and nodes are defined by the size of the circles with colors representing directionality of differential expression in the vmPFC (depicted in the bottom left panel). c, RRHO map directly comparing transcriptional profiles of females after DUSP6 downregulation with 21 days of CVS in the female vmPFC. Degree of significance is depicted in the color bar below the RRHO map. d, Top ontological terms enriched for downregulated genes following DUSP6 downregulation in female vmPFC. e, Venn diagram displaying the overlap between genes differentially expressed (p<0.05) in females after 21 days of CVS (yellow) and females after DUSP6 downregulation (blue) in the vmPFC. Heatmaps on the right compare transcriptional changes (log fold change; right to the heatmaps) in females after 21 days of CVS and females after DUSP6 downregulation in the vmPFC.

Article Snippet: DUSP6 and EMX1 coding regions were excised from DUSP6 (Origene cat# MR222688) and EMX1 (Origene cat# RC208006) expression clones and cloned into a p1005+ HSV vector to induce overexpression in the brain.

Techniques: Expressing

Journal: Journal of Ginseng Research

Article Title: Ginsenoside Rk1 ameliorates paracetamol-induced hepatotoxicity in mice through inhibition of inflammation, oxidative stress, nitrative stress and apoptosis

doi: 10.1016/j.jgr.2017.07.003

Figure Lengend Snippet: Pretreatment with Rk1 protected against APAP-induced liver injury. (A) 4-hydroxynonenal (4-HNE), (B) cytochrome P450 E1 (CYP2E1), and (C) 3-nitrotyrosine (3-NT). The expression levels of 4-HNE, CYP2E1 (green) and 3-NT (red) in tissue sections isolated from different groups were assessed by immunofluorescence. Representative immunofluorescence images were taken at 200×. 4′, 6-diamidino-2-phenylindole (DAPI) (blue) was used as a nuclear counterstain. All data are expressed as the mean ± standard deviation, n = 8. ** p < 0.01 versus normal group; ## p < 0.01 versus APAP group. APAP, paracetamol.

Article Snippet: The antibodies, including rabbit monoclonal anti-iNOS, anti-COX-2, anti-Bax, anti-Bcl-2, anti-cytochrome P450 E1 (CPY2E1), anti-4-HNE, and anti-3-nitrotyrosine (3-NT), were provided by BOSTER Biological Technology (Wuhan, China) or Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, Isolation, Immunofluorescence, Standard Deviation