mkp 1 sirna  (Santa Cruz Biotechnology)

Journal: Journal of Neuroinflammation

Article Title: 5, 8, 11, 14-eicosatetraynoic acid suppresses CCL2/MCP-1 expression in IFN-γ-stimulated astrocytes by increasing MAPK phosphatase-1 mRNA stability

doi: 10.1186/1742-2094-9-34

Figure Lengend Snippet: ETYA acts in a PPAR-α-independent manner to suppress CCL2/MCP-1 expression through induction of MKP-1 expression and phosphatase activity . (A and B) ETYA- or WY-14643-treated astrocytes were stimulated with IFN-γ for 2 h. MKP-1 protein levels and JNK phosphorylation were analyzed by Western blotting ( A ), and MKP-1 and CCL2/MCP-1 transcript levels were determined by qRT-PCR ( B ). (C and D) Cell lysates immunoprecipitated with an anti-MKP-1 antibody were incubated with p -NPP for 4 h and then analyzed spectrophotometrically at 405 nm ( C ), or incubated with lysates from IFN-γ-activated astrocytes followed by Western blot analysis of eluates ( D ). (E-I) Primary astrocytes were transfected with an MKP-1-specific ( E and F ) or PPAR-α-specific siRNA duplex (G-I) or a nonsilencing control siRNA. Forty-eight h after transfection, cells were stimulated with IFN-γ for 2 h or 12 h in the absence or presence of ETYA. MKP-1 protein levels and phospho-JNK, and transcript levels of MKP-1 and CCL2/MCP-1 were determined by Western blot analysis ( E and G ) and qRT-PCR ( F and H ). MCP-1 protein secretion was analyzed by ELISA ( I ). Efficiency of siRNA-mediated PPAR-α silencing was demonstrated by monitoring expression of acetyl CoA synthase (ACS), a PPAR-α-dependent gene (data not shown). Values are means ± SDs. of three independent experiments. * p < 0.05, ** p < 0.01, NS; non significant.

Article Snippet: siRNA duplex oligonucleotides targeting MKP-1 (5'-CCA ATT GTC CTA ACC ACT T-3'), CB1 (5'-CGA AGG UGA CCA UGU CUG UTT-3'), HuR (5'-GAU GCC AAC UUG UAC AUC ATT-3') and PPAR-α (5'-GGC UAA AGC UGG CGU ACG AUU-3') were chemically synthesized by Bioneer (Korea).

Techniques: Expressing, Activity Assay, Western Blot, Quantitative RT-PCR, Immunoprecipitation, Incubation, Transfection, Enzyme-linked Immunosorbent Assay

Journal: Journal of Neuroinflammation

Article Title: 5, 8, 11, 14-eicosatetraynoic acid suppresses CCL2/MCP-1 expression in IFN-γ-stimulated astrocytes by increasing MAPK phosphatase-1 mRNA stability

doi: 10.1186/1742-2094-9-34

Figure Lengend Snippet: ETYA suppresses CCL2/MCP-1 expression by inducing MKP-1 expression in brain microglia . (A - C) Primary microglia were stimulated with 10 U/ml IFN-γ for 2 h or 12 h in the presence of ETYA or WY-14643. MKP-1 protein expression and JNK phosphorylation were analyzed by Western blotting ( A ), and MKP-1 and CCL2/MCP-1 transcript levels and protein release were examined by qRT-PCR ( B ) and ELISA ( C ). (D - G) Rat microglia were transfected with a siRNA duplex specific for MKP-1 ( D and E ) or PPAR-α ( F and G ). After 48 h, cells were stimulated with IFN-γ for 2 h in the presence or absence of ETYA. MKP-1 protein and JNK phosphorylation were analyzed by Western blotting ( D and F ), and MKP-1 and CCL2/MCP-1 transcript levels were examined by RT-PCR ( E and G ). Values are means ± SDs of three independent experiments. * p < 0.05, ** p < 0.01, NS; non significant.

Article Snippet: siRNA duplex oligonucleotides targeting MKP-1 (5'-CCA ATT GTC CTA ACC ACT T-3'), CB1 (5'-CGA AGG UGA CCA UGU CUG UTT-3'), HuR (5'-GAU GCC AAC UUG UAC AUC ATT-3') and PPAR-α (5'-GGC UAA AGC UGG CGU ACG AUU-3') were chemically synthesized by Bioneer (Korea).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Transfection, Reverse Transcription Polymerase Chain Reaction

Journal: Journal of Neuroinflammation

Article Title: 5, 8, 11, 14-eicosatetraynoic acid suppresses CCL2/MCP-1 expression in IFN-γ-stimulated astrocytes by increasing MAPK phosphatase-1 mRNA stability

doi: 10.1186/1742-2094-9-34

Figure Lengend Snippet: The ETYA-induced increase in MKP-1 expression involves a HuR-mediated increase in MKP-1 mRNA stability resulting from HuR cytoplasmic translocation . (A) Astrocytes were treated with Act D for the indicated periods in the presence of ETYA, after which MKP-1 mRNA levels were determined by conventional RT-PCR (left, upper panel) and qRT-PCR (right panel). The bar graph represents the intensities of MKP-1 bands normalized against those of GAPDH (left, bottom panel). All data are presented as means ± SDs of three independent experiments (* p < 0.05). (B and C) Astrocytes were transfected with a HuR-specific or nonsilencing control siRNA. Forty-eight h after transfection, cells were stimulated with IFN-γ in the absence or presence of ETYA. MKP-1 mRNA and protein levels were determined by RT-PCR and Western blot analysis (B), and MKP-1 mRNA stability was determined by qRT-PCR ( C ). The data are presented as means ± SDs of three independent experiments (* p < 0.05 versus IFN-γ + ETYA + con siRNA group). (D) Astrocytes were stimulated with IFN-γ in the presence or absence of ETYA, and nuclear extracts (NE) and cytosolic extracts (CE) were prepared for Western blotting. (E) Confocal microscopic images of astrocytes immunostained with antibodies against HuR, GFAP (astrocyte marker), and DAPI (nuclear marker), under the indicated conditions. Insets are magnified images of the corresponding boxed regions. Scale bars, 20 μm.

Article Snippet: siRNA duplex oligonucleotides targeting MKP-1 (5'-CCA ATT GTC CTA ACC ACT T-3'), CB1 (5'-CGA AGG UGA CCA UGU CUG UTT-3'), HuR (5'-GAU GCC AAC UUG UAC AUC ATT-3') and PPAR-α (5'-GGC UAA AGC UGG CGU ACG AUU-3') were chemically synthesized by Bioneer (Korea).

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

Journal: Journal of Neuroinflammation

Article Title: 5, 8, 11, 14-eicosatetraynoic acid suppresses CCL2/MCP-1 expression in IFN-γ-stimulated astrocytes by increasing MAPK phosphatase-1 mRNA stability

doi: 10.1186/1742-2094-9-34

Figure Lengend Snippet: A model showing the anti-inflammatory mechanism of ETYA in IFN-γ-stimulated glial cell . IFN-γ induces JNK/Jun phosphorylation and increases CCL2/MCP-1 gene expression. ETYA, but not fibrates, increases MKP-1 mRNA stability by inducing HuR cytoplasmic shuttling, and thereby suppresses IFN-γ-induced JNK signaling and CCL2/MCP-1 expression.

Article Snippet: siRNA duplex oligonucleotides targeting MKP-1 (5'-CCA ATT GTC CTA ACC ACT T-3'), CB1 (5'-CGA AGG UGA CCA UGU CUG UTT-3'), HuR (5'-GAU GCC AAC UUG UAC AUC ATT-3') and PPAR-α (5'-GGC UAA AGC UGG CGU ACG AUU-3') were chemically synthesized by Bioneer (Korea).

Techniques: Expressing

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Hypoxia Downregulates MAPK/ERK but Not STAT3 Signaling in ROS-Dependent and HIF-1-Independent Manners in Mouse Embryonic Stem Cells

doi: 10.1155/2017/4386947

Figure Lengend Snippet: Primers, appropriate annealing temperatures, and PCR product lengths.

Article Snippet: Cells were transfected by commercially available siRNA against DUSP1 (sc-35938), DUSP5 (sc-60555), DUSP6 (sc-39001) transcripts (each consisting of a pool of 3 target-specific 19-25 nt siRNAs designed to knock down gene expression), or related nonsilencing control (all Santa Cruz Biotechnology, USA) using Lipofectamine RNAiMAX Reagents (Thermo Fisher Scientific Inc., USA) according to the manufacturer's instructions.

Techniques:

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Hypoxia Downregulates MAPK/ERK but Not STAT3 Signaling in ROS-Dependent and HIF-1-Independent Manners in Mouse Embryonic Stem Cells

doi: 10.1155/2017/4386947

Figure Lengend Snippet: Relative expressions of DUSP1 (a), DUSP5 (b), and DUSP6 (c) in hypoxia in wild-type and HIF-1 α −/− ES cells. Statistical significance was determined by T test ( ∗ P < 0.05). Effect of siRNA-mediated silencing on DUSP1 (d), DUSP5 (e), and DUSP6 (f) expression in wild-type ES cells determined by qRT-PCR. Data are presented as mean + SEM from at least three independent experiments. Statistical significance was determined by one-way analysis of variance ANOVA and post hoc Bonferroni's multiple comparison test ( ∗ P < 0.05). Effect of DUSP1, 5, and 6 siRNAs on ERK phosphorylation and DUSP6 levels in wild-type ES cells (g). The total level of β -actin was used as a loading control. Densitometry analysis of western blots expressed as fold change in DUSP6 protein expression (h) or ERK phosphorylation (i). Data are presented as mean + SEM from at least three independent experiments. Statistical significance was determined by one-way analysis of variance ANOVA and post hoc Bonferroni's multiple comparison test ( ∗ P < 0.05).

Article Snippet: Cells were transfected by commercially available siRNA against DUSP1 (sc-35938), DUSP5 (sc-60555), DUSP6 (sc-39001) transcripts (each consisting of a pool of 3 target-specific 19-25 nt siRNAs designed to knock down gene expression), or related nonsilencing control (all Santa Cruz Biotechnology, USA) using Lipofectamine RNAiMAX Reagents (Thermo Fisher Scientific Inc., USA) according to the manufacturer's instructions.

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Molecular Pain

Article Title: Mitogen activated protein kinase phosphatase-1 prevents the development of tactile sensitivity in a rodent model of neuropathic pain

doi: 10.1186/1744-8069-8-34

Figure Lengend Snippet: MKP-1 expression in spinal cord following peripheral nerve injury. Quantification of MKP-1 protein ( a ) and representative Western blot of MKP-1 expression ( b ) in L5–L6 spinal cord of naïve rats and of rats that have undergone sham (Sh) or L5 nerve transection (L5NT or NT) surgery ( n = 3–6) on postoperative days 1 (D1) and 4 (D4) . * P < 0.05 for sham versus naïve on postoperative days 1 and 4, also between sham and L5 nerve transection groups on postoperative day 4. All data are expressed as mean ± s.e.m.

Article Snippet: Stealth MKP-1 siRNAs (0.1, 1, and 10 nM) were transfected for 24 hr in BV-2 stably expressing MKP-1 using RNAi max transfection reagent (Invitrogen, San Diego, CA), following manufacturer’s instructions.

Techniques: Expressing, Western Blot

Journal: Molecular Pain

Article Title: Mitogen activated protein kinase phosphatase-1 prevents the development of tactile sensitivity in a rodent model of neuropathic pain

doi: 10.1186/1744-8069-8-34

Figure Lengend Snippet: In vitro characterization of rat MKP-1 overexpression. MKP-1 mRNA levels ( a , qRT-PCR) and Western blot of MKP-1 protein ( b ) in normal BV-2 cells and stably MKP-1 overexpressing BV-2 cells. ( c ) Representative Western blots of p-p38, and p-JNK expression in BV-2 cells stably overexpressing MKP-1 following 1 or 2 h incubation in medium (control alone) or lipopolysaccharide (LPS) stimulation (1 μg/ml). IL-6 ( d ), TNF-α ( e ), nitrite ( f ), and MCP-1) ( g ) release in control (normal) and stably expressing MKP-1 BV-2 cells challenged with LPS (1 μg/ml) for 6 or 12 h. Results are expressed as mean ± s.e.m. of three experiments in triplicate. * P < 0.05 vs. control BV-2 cells.

Article Snippet: Stealth MKP-1 siRNAs (0.1, 1, and 10 nM) were transfected for 24 hr in BV-2 stably expressing MKP-1 using RNAi max transfection reagent (Invitrogen, San Diego, CA), following manufacturer’s instructions.

Techniques: In Vitro, Over Expression, Quantitative RT-PCR, Western Blot, Stable Transfection, Expressing, Incubation

Journal: Molecular Pain

Article Title: Mitogen activated protein kinase phosphatase-1 prevents the development of tactile sensitivity in a rodent model of neuropathic pain

doi: 10.1186/1744-8069-8-34

Figure Lengend Snippet: Reversal effects of BV-2 cell MKP-1 induction in cytokine production by siRNA MKP-1 knockdown. ( a ) Efficient siRNA BV-2 cell transfection using Alexa-555 red fluorescent-labeled siRNA. ( b ) MKP-1 mRNA level in BV-2 cells stably expressing MKP-1 after 24 h transfection with MKP-1 siRNA compared to negative control siRNA and vehicle treated cells. IL-6 ( c ), TNF-α ( d ), and IL-1β ( e ) mRNA quantification (qRT-PCR) in control (normal) and stably expressing MKP-1 BV-2 cells transfected 24 h with a negative siRNA, or a siRNA against MKP-1 and challenged with LPS (1 μg/ml) for 6 h (IL-6 and TNF-α) or 12 h (IL-1β). Since MKP-1 siRNA at 10 nM displayed an opposite effect than expected in TNF-α mRNA (potential non-specific effect), 1 nM was used for protein release experiments. Monocyte chemoattractant protein (MCP)-1 ( f ), IL-6 ( g ), TNF-α ( h ), and nitrite ( i ) release in control (normal) and stably expressing MKP-1 BV-2 cells transfected 24 h with a negative siRNA, or a siRNA against MKP-1 and challenged with LPS (1 μg/ml) for 6 h (MCP-1, IL-6, and TNF-α) or 12 h (nitrite). Results are expressed as mean ± s.e.m. of three experiments in duplicate. * P < 0.05 vs. BV-2 cells stably expressing MKP-1 (pMKP1 +).

Article Snippet: Stealth MKP-1 siRNAs (0.1, 1, and 10 nM) were transfected for 24 hr in BV-2 stably expressing MKP-1 using RNAi max transfection reagent (Invitrogen, San Diego, CA), following manufacturer’s instructions.

Techniques: Transfection, Labeling, Stable Transfection, Expressing, Negative Control, Quantitative RT-PCR

Journal: Molecular Pain

Article Title: Mitogen activated protein kinase phosphatase-1 prevents the development of tactile sensitivity in a rodent model of neuropathic pain

doi: 10.1186/1744-8069-8-34

Figure Lengend Snippet: In vivo induction of spinal MKP-1. Quantification of MKP-1 mRNA ( n = 3) ( a ) and of MKP-1 protein ( n = 3) ( b ), as well as a representative Western blot of MKP-1 expression ( c ), in L5–L6 spinal cord in rats that have undergone L5 nerve transection surgery and been treated i.t. with PEI only, PEI + empty cDNA, and PEI + MKP-1 cDNA four days after treatment and surgery. * P < 0.05 versus PEI only or PEI + empty vector. All data are expressed as mean ± s.e.m. ( d ) Representative images obtained by confocal microscopy and immunofluorescence depicting MKP-1 (in green) and the microglial cell marker Iba-1 (in red), the neuronal marker NeuN (in red), and the astrocytic cell markers GFAP (in red) and S100b (in red) in spinal cord of rats four days after L5 nerve transection and treated with PEI + MKP-1 cDNA or PEI only. Arrows indicate co-regionalization (in yellow/orange). Arrowheads show punctate co-regionalization in NeuN-expressing neurons.

Article Snippet: Stealth MKP-1 siRNAs (0.1, 1, and 10 nM) were transfected for 24 hr in BV-2 stably expressing MKP-1 using RNAi max transfection reagent (Invitrogen, San Diego, CA), following manufacturer’s instructions.

Techniques: In Vivo, Western Blot, Expressing, Plasmid Preparation, Confocal Microscopy, Immunofluorescence, Marker

Journal: Molecular Pain

Article Title: Mitogen activated protein kinase phosphatase-1 prevents the development of tactile sensitivity in a rodent model of neuropathic pain

doi: 10.1186/1744-8069-8-34

Figure Lengend Snippet: Effects of in vivo induction of spinal MKP-1 in behavioral hypersensitivity and spinal p-p38 expression. Withdrawal threshold to mechanical stimulation ( a ) in rats undergone L5 nerve transection surgery and treated i.t. with PEI only ( n = 9), PEI + empty cDNA ( n = 4), and PEI + MKP-1 cDNA ( n = 9) four days after treatment and surgery. Quantification of p-p38 protein ( b ) and representative Western blot of p-p38 ( c ) expression from L5–L6 spinal cord of rats that have undergone L5 nerve transection surgery and been treated i.t. with PEI only ( n = 3), PEI + empty cDNA ( n = 3) and PEI + MKP-1 cDNA ( n = 3) four days after treatment and surgery. * P < 0.05 versus PEI only or PEI + empty vector. All data are expressed as mean ± s.e.m. ( d ) Representative images obtained by confocal microscopy and immunofluorescence depicting p-p38 (in green) and the microglial marker Iba-1 (in red), the neuronal marker NeuN (in red), and the astrocytic marker S100b (in red) in spinal cord of rats four days after L5 nerve transection and treated with PEI + MKP-1 cDNA or PEI only. Arrows indicate co-regionalization (in yellow/orange).

Article Snippet: Stealth MKP-1 siRNAs (0.1, 1, and 10 nM) were transfected for 24 hr in BV-2 stably expressing MKP-1 using RNAi max transfection reagent (Invitrogen, San Diego, CA), following manufacturer’s instructions.

Techniques: In Vivo, Expressing, Western Blot, Plasmid Preparation, Confocal Microscopy, Immunofluorescence, Marker

Journal: Molecular Pain

Article Title: Mitogen activated protein kinase phosphatase-1 prevents the development of tactile sensitivity in a rodent model of neuropathic pain

doi: 10.1186/1744-8069-8-34

Figure Lengend Snippet: Effects of in vivo induction of spinal MKP-1 in pro-inflammatory/algesic effectors. Concentrations of IL-1β ( a ), IL-6 ( b ), TNF-α ( c ), and MCP-1 ( d ) from L5–L6 spinal cord of rats undergone L5 nerve transection surgery and treated i.t. with PEI only ( n = 5), PEI + empty cDNA ( n = 4), and PEI + MKP-1 cDNA ( n = 9) four days after treatment and surgery. * P < 0.05 versus PEI only or PEI + empty vector. All data are expressed as mean ± s.e.m.

Article Snippet: Stealth MKP-1 siRNAs (0.1, 1, and 10 nM) were transfected for 24 hr in BV-2 stably expressing MKP-1 using RNAi max transfection reagent (Invitrogen, San Diego, CA), following manufacturer’s instructions.

Techniques: In Vivo, Plasmid Preparation

Journal: PLoS ONE

Article Title: The Polyphenol Fisetin Protects Bone by Repressing NF-κB and MKP-1-Dependent Signaling Pathways in Osteoclasts

doi: 10.1371/journal.pone.0068388

Figure Lengend Snippet: (A). Raw264.7 osteoclast precursors were pre-incubated with DMSO as control (−) or fisetin (5 µM) for 3 hours, then induced for 5 to 60 minutes with RANKL and DMSO as control (−) or fisetin (5 µM). Total protein extracts were analyzed by western-blotting for the indicated proteins. MKP-1 exp+ is a long exposure blot that highlights the difference of signal intensities at T15 min (*). Blots are representative of 3 independent experiments. (B). Raw264.7 were incubated with fisetin (5 µM) for 15 minutes to 8 hours, and total protein extracts were analyzed by western-blotting for the indicated proteins. Blots are representative of 3 independent experiments. (C). Raw264.7 were incubated with fisetin (5 µM) for 30 minutes to 8 hours, and MKP-1 mRNA was analyzed by RT qPCR. (*) significantly different from T0, p<0.05. (n = 3 wells, representative of 3 independent experiments). (D). Raw264.7 were incubated with the proteasome inhibitor MG132 (25 µM) for 30 minutes to 8 hours, and total protein extracts were analyzed by western-blotting for the indicated proteins. Blots are representative of 3 independent experiments. (E). Raw264.7 cells were transfected with Myc-MKP-1 and HA-Ub and incubated with DMSO as control (fisetin 0 µM) or fisetin (5 µM) for 4 hours. Whole cell extracts were prepared as described in the section on ubiquitination assays (“ ”) and were subjected to immunoprecipitation (IP) with anti-Myc prior western-blotting analysis. Blots are representative of 3 independent experiments. (F). shControl (shCtrl) and shMKP-1 Raw264.7 were pre-incubated with DMSO as control (fisetin 0 µM) or fisetin (5 µM) for 3 hours, then induced for 15 minutes with RANKL and DMSO as control (fisetin 0 µM) or fisetin (5 µM). Total protein extracts were analyzed by western-blotting for the indicated proteins. Blots are representative of 3 independent experiments. (G). shCtrl and shMKP-1 Raw264.7 were pre-incubated with DMSO as control (fisetin 0 µM) or fisetin (5 µM) for 3 hours, then induced for 4 days with RANKL and DMSO as control (fisetin 0 µM) or fisetin (5 µM) and the indicated mRNAs were analyzed by RT qPCR. (n = 3 wells, representative of 3 independent experiments). (H). Similar experiments as in G were conducted and TRAP staining was performed at the end of the differentiation process. Scale bars correspond to 250 µm. (n = 3 wells, representative of 3 independent experiments).

Article Snippet: Control and MKP-1 shRNA vectors were stably introduced in Raw264.7 using lentiviral particules (Sigma-Aldrich) followed by puromycin selection (4 µg/ml).

Techniques: Incubation, Western Blot, Quantitative RT-PCR, Transfection, Immunoprecipitation, Staining

Journal: PLoS ONE

Article Title: The Polyphenol Fisetin Protects Bone by Repressing NF-κB and MKP-1-Dependent Signaling Pathways in Osteoclasts

doi: 10.1371/journal.pone.0068388

Figure Lengend Snippet: Fisetin negatively controls osteoclasts differentiation process by inhibiting RANKL-induced NF-κB signaling (a), stabilizing MKP-1 (b), the phosphatase that negatively controls p38 MAPK and JNK signaling pathways, thus inhibiting their RANKL-induced activation (b’) and counteracting the RANKL-induced c-Fos expression (c). These actions result in a transcriptional repression of the key transcription factor NFATc1 and a subsequent inhibition of its target genes: CTR, TRAP, MMP9 or cathepsin K.

Article Snippet: Control and MKP-1 shRNA vectors were stably introduced in Raw264.7 using lentiviral particules (Sigma-Aldrich) followed by puromycin selection (4 µg/ml).

Techniques: Activation Assay, Expressing, Inhibition