phospho-jnk Search Results


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  • 99
    Cell Signaling Technology Inc phospho jnk
    Dabrafenib reduced the expression of phosphorylated <t>JNK</t> and c-jun in both SH-SY5Y cells and mice ( A ) LDH/cell viability assay under the condition of <t>ERK</t> inhibitor. Neuroprotective effects of dabrafenib (5 μM) were not dependent on the downregulation of activated ERK expression by PD98059 (30 μM) or U0126 (3 μM). Neuroprotective effects were assessed by the LDH and cell viability assay in SH-SY5Y cells at 24 h after MPP + (3 mM) exposure. ** , P -value
    Phospho Jnk, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 8342 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc anti phospho jnk
    Ran(K152A) enhances the transformed phenotype exhibited by SKBR3 breast cancer cells. A , A single SKBR3 cell clone stably expressing either the vector-alone, wild-type Ran, or Ran(K152A) were subjected to anchorage-independent growth (soft agar) assays. The experiments were performed three times and the resulting colonies that formed in each experiment were counted, averaged together, and graphed. The error bars represent standard deviation. Representative images of the soft agar assay are shown. B , SKBR3 cells stably expressing the vector-alone, wild-type Ran, or Ran(K152A) were serum-starved overnight and lysed. As a positive control, an additional plate of serum-starved SKBR3 cells expressing the vector alone was stimulated with medium containing serum for 10 min prior to being lysed. The cell extracts were analyzed by Western blot analysis using <t>phospho-ERK,</t> <t>phospho-JNK,</t> and HA antibodies. Similar results were obtained using additional clones (data not shown).
    Anti Phospho Jnk, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 2839 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc p jnk
    Light exposure increased the expression of p-MAPKs in the retina. MAPKs activation in retina, represented by the levels of p-ERK1/2, <t>p-JNK</t> and <t>p-P38,</t> were detected with immunofluorescence. The green arrows points to the ONL, and the yellow arrows points to the MAPKs enhancement area after light exposure. p-ERK1/2 (a), p-JNK (b) and p-P38 (c) were elevated in the rat model of light-induced retinal degeneration, mainly in the ONL, on days 3 and 5 when compared with their levels in the normal retina. MAPK: Mitogen-activated protein kinase; p-ERK1/2: Phosphorylated-extracellular regulated kinase 1/2; JNK: c-Jun N-terminal kinase; ONL: Outer nuclear layer; INL: Inner nuclear layer; GCL: Ganglion cell layer.
    P Jnk, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 7240 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology phospho jnk
    Rosiglitazone counteracts <t>JNK-induced</t> β-cell dysfunction. GTT ( A ) and the corresponding area under the curve (AUC) for the first 60 min ( B ), glucose-stimulated insulin secretion ( C ), the corresponding AUC for the first 15 min ( D ), and ITT ( E ) performed in control and MKK7D 2-month-old mice treated for 10 days with rosiglitazone (Rosi) or PBS as indicated. F : Insulin secretion of pancreatic islets isolated from control and MKK7D mice treated with PBS or rosiglitazone. Islets were stimulated with glucose and insulin as indicated. G : Immunoblot analysis performed in extracts of pancreatic islets from control and MKK7D mice treated with rosiglitazone and insulin (Ins) (20 min) as indicated. The ratios of <t>phospho-Akt</t> (P-Akt) and phospho-JNK (P-JNK) to AKT and JNK, respectively, are represented in the right panel . Assays were performed with at least 10 animals per group and repeated at least 3 times. * P
    Phospho Jnk, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 644 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc phospho jnk1 2
    TBMS1 inhibits the phosphorylation of p38 and ERK1/2 in LPS-exposed BV-2 cells. The BV-2 cells were pretreated for 1 h with TBMS1 (1, 2 and 4 μM), then incubated with LPS (1 μg/mL) for 1 h. ( A ) The protein levels of p38, ERK1/2, <t>JNK1/2</t> and their phosphorylated forms were tested by western blotting. The phosphorylation of p38 ( B ), ERK1/2 ( C ) and JNK1/2 ( D ) was analyzed relative to β-actin. The results are shown as mean ± SD of three independent experiments. # p
    Phospho Jnk1 2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 467 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology anti phospho jnk
    Neuroprotection in hippocampal CA3 neurons, and the prevention of <t>p-JNK,</t> <t>BACE1</t> and NOX2 induction by early continuous 17β-oestradiol (E2) replacement for 10 weeks in rats with LTED. ( A ) Representative staining of NeuN, TUNEL and amyloid-β1–42 of CA3 region from sham (Sh), and animals treated with 17β-oestradiol or placebo (Pla) immediately after ovariectomy for the entire 10-week period. The rats were subjected to sham or 10 min ischaemia followed by 7 days of reperfusion. ( B ) Cell-counting study shows the number of surviving neurons (% changes of sham) and TUNEL-positive cells per 250 µm length of medial CA3 region. Magnification: ×40. Scale bar = 50 µm. n = 6–7 in each group. # P
    Anti Phospho Jnk, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 276 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti phosphorylated jnk
    Effects of CoPP treatment on the protein levels of Nrf2, HO-1, <t>NQO1</t> and <t>JNK</t> in the sciatic nerve of diabetic mice. The protein levels of Nrf2 ( A ), HO-1 ( B ), NQO1 ( C ) and JNK ( D ) in the sciatic nerve from db/db mice treated with CoPP or vehicle and in db/+ mice treated with vehicle are represented. For each protein, * indicates significant differences as compared to db/+ mice treated with vehicle and # indicates significant differences vs. db/db mice treated with CoPP ( p
    Anti Phosphorylated Jnk, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 221 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Cell Signaling Technology Inc rabbit anti phospho sapk jnk
    Effects of GSI treatment on MAPK, PI3K/AKT and NF-κB pathway activation in LPS/IC-activated macrophages. (A-B) IFNγ-primed BMMs were pre-treated with vehicle control DMSO or GSI (25 μM) for 30 min and activated by LPS/ICs for 5, 15, 30 and 60 min. Phospho-p38, p38 phospho-p44-42, p44-42, <t>phospho-SAPK/JNK,</t> SAPK/JNK, phospho-Akt, Akt and the loading control β-actin were detected by Western blotting. Representative data from 1 of 3 independent experiments are shown. (C) IFNγ-primed BMMs were activated by LPS/ICs for 4 hrs in the presence of vehicle control DMSO or GSI (25 μM). NF-κB p50 was detected by immunofluorescence staining. The arrows indicate cells with decreased or no p50 nuclear translocation (green). Representative data from 1 of 2 independent experiments are shown.
    Rabbit Anti Phospho Sapk Jnk, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 89/100, based on 209 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Dabrafenib reduced the expression of phosphorylated JNK and c-jun in both SH-SY5Y cells and mice ( A ) LDH/cell viability assay under the condition of ERK inhibitor. Neuroprotective effects of dabrafenib (5 μM) were not dependent on the downregulation of activated ERK expression by PD98059 (30 μM) or U0126 (3 μM). Neuroprotective effects were assessed by the LDH and cell viability assay in SH-SY5Y cells at 24 h after MPP + (3 mM) exposure. ** , P -value

    Journal: Human Molecular Genetics

    Article Title: In silico drug screening by using genome-wide association study data repurposed dabrafenib, an anti-melanoma drug, for Parkinson’s disease

    doi: 10.1093/hmg/ddy279

    Figure Lengend Snippet: Dabrafenib reduced the expression of phosphorylated JNK and c-jun in both SH-SY5Y cells and mice ( A ) LDH/cell viability assay under the condition of ERK inhibitor. Neuroprotective effects of dabrafenib (5 μM) were not dependent on the downregulation of activated ERK expression by PD98059 (30 μM) or U0126 (3 μM). Neuroprotective effects were assessed by the LDH and cell viability assay in SH-SY5Y cells at 24 h after MPP + (3 mM) exposure. ** , P -value

    Article Snippet: Antibodies against caspase-3 (#9665, 1:1000), cleaved caspase-9 (#7237, 1:1000), ERK 1/2 (#4695, 1:1000), phospho-ERK 1/2 (#4370, 1:2000), JNK (#9252, 1:1000), phospho-JNK (#4668, 1:1000), phospho-c-jun (#3270, 1:1000) and phospho-B-Raf (Ser445; #2696, 1:1000) were all purchased from Cell Signaling Technology (Beverly, MA, USA).

    Techniques: Expressing, Mouse Assay, Viability Assay

    JNK inhibits Bim expression through suppressing Smurf2-mediated degradation of Ezh2. a mRNA (left) and protein (right) levels of Ezh2 in human erythroblasts triggered by SP600125 assessed by q-PCR or immunoblotting. b Ezh2 protein level induced by JNK1 silencing measured by immunoblotting. c Time course of Ezh2 levels in CHX-treated human erythroblasts with DMSO or SP600125. d SP600125-induced degradation of Ezh2 in the presence of DMSO or MG132 (10 µM) by immunoblotting. e mRNA expression of HOXA9 in human erythroblasts induced by SP600125 (left) or JNK1 shRNA (right) by q-PCR. f Quantitative chromatin immunoprecipitation analysis of occupancy of Ezh2 (left, n = 5) and H3K27me3 (right, n = 3) on Bim promoter in human erythroblasts treated with vehicle or SP600125. g mRNA (left) and protein (right) levels of Bim induced by GSK126 (5 µM) for 12 h. h Apoptosis of human erythroblasts trigged by GSK126 and measured by flow cytometry. i mRNA expression of Bim induced by Ezh2 silencing. j Representative flow cytometry profile (left) and quantification (right) of apoptotic human erythroblasts induced by Ezh2-specific shRNA ( n = 4). k Ezh2 protein levels in P38α +/ − and P38α − / − erythroblasts by immunoblotting. l mRNA levels of HOXA9 in P38α +/ − and P38α − / − erythroblasts by q-PCR. m Apoptosis in P38α +/ − and P38α − / − erythroblasts subjected to GSK126 (5 µM) ( n = 4). n Protein levels of Ezh2, Smurf2, and Bim in human erythroblasts transduced with control or smurf2-specific shRNA then treated with SP600125. o Representative flow cytometry profile (left) and quantification (right) of SP600125-induced apoptosis of human erythroblasts transduced with control or smurf2-specific shRNA ( n = 3). Blots are representative of two independent experiments. Data are shown as mean ± s.e.m. * P

    Journal: Nature Communications

    Article Title: P38α/JNK signaling restrains erythropoiesis by suppressing Ezh2-mediated epigenetic silencing of Bim

    doi: 10.1038/s41467-018-05955-2

    Figure Lengend Snippet: JNK inhibits Bim expression through suppressing Smurf2-mediated degradation of Ezh2. a mRNA (left) and protein (right) levels of Ezh2 in human erythroblasts triggered by SP600125 assessed by q-PCR or immunoblotting. b Ezh2 protein level induced by JNK1 silencing measured by immunoblotting. c Time course of Ezh2 levels in CHX-treated human erythroblasts with DMSO or SP600125. d SP600125-induced degradation of Ezh2 in the presence of DMSO or MG132 (10 µM) by immunoblotting. e mRNA expression of HOXA9 in human erythroblasts induced by SP600125 (left) or JNK1 shRNA (right) by q-PCR. f Quantitative chromatin immunoprecipitation analysis of occupancy of Ezh2 (left, n = 5) and H3K27me3 (right, n = 3) on Bim promoter in human erythroblasts treated with vehicle or SP600125. g mRNA (left) and protein (right) levels of Bim induced by GSK126 (5 µM) for 12 h. h Apoptosis of human erythroblasts trigged by GSK126 and measured by flow cytometry. i mRNA expression of Bim induced by Ezh2 silencing. j Representative flow cytometry profile (left) and quantification (right) of apoptotic human erythroblasts induced by Ezh2-specific shRNA ( n = 4). k Ezh2 protein levels in P38α +/ − and P38α − / − erythroblasts by immunoblotting. l mRNA levels of HOXA9 in P38α +/ − and P38α − / − erythroblasts by q-PCR. m Apoptosis in P38α +/ − and P38α − / − erythroblasts subjected to GSK126 (5 µM) ( n = 4). n Protein levels of Ezh2, Smurf2, and Bim in human erythroblasts transduced with control or smurf2-specific shRNA then treated with SP600125. o Representative flow cytometry profile (left) and quantification (right) of SP600125-induced apoptosis of human erythroblasts transduced with control or smurf2-specific shRNA ( n = 3). Blots are representative of two independent experiments. Data are shown as mean ± s.e.m. * P

    Article Snippet: Antibodies against P38α (9212,1:1000), phospho-P38 (9211, 1:1000), JNK1(3708, 1:1000), Total JNK (9252, 1:1000), phosphor-JNK (9251,1:1000), Ezh2 (5246, 1:3000), LRF (13097, 1:1000), CDK1(28439, 1:1000), phosphor-CDK1(9111, 1:1000), P53 (2524, 1:1000), Myc tag (2272, 1:1000), and phosphor-T-P (9391, 1:1000) are from Cell Signaling.

    Techniques: Expressing, Polymerase Chain Reaction, shRNA, Chromatin Immunoprecipitation, Flow Cytometry, Cytometry, Transduction

    JNK-mediated Cdk1 inhibition stabilizes Ezh2 and supports erythroblasts. a , b Tyr-15 phosphorylation of Cdk1 in SP600125-treated ( a ) or in JNK1-silenced ( b ) human erythroblasts by immunoblotting. c Phosphorylation of Tyr-15 of Cdk1 in P38α +/ − and P38α − / − erythroblasts. d – f Human erythroblasts transduced with control or cdk1-specific shRNA and treated with vehicle or SP600125. Protein levels of Ezh2 and Cdk1 by immunoblotting ( d ), mRNA expression of Bim by q-PCR ( e ), and representative flow cytometry profile (left) and quantification (right) of apoptotic human erythroblasts ( f ) ( n = 4). g Schematic outlining a proposed mechanism mediating rewired JNK activation due to lack of P38 via Map3k4 and their downstream targets regulating erythropoiesis. Blots are representative of three independent experiments. Data are shown as mean ± s.e.m. * P

    Journal: Nature Communications

    Article Title: P38α/JNK signaling restrains erythropoiesis by suppressing Ezh2-mediated epigenetic silencing of Bim

    doi: 10.1038/s41467-018-05955-2

    Figure Lengend Snippet: JNK-mediated Cdk1 inhibition stabilizes Ezh2 and supports erythroblasts. a , b Tyr-15 phosphorylation of Cdk1 in SP600125-treated ( a ) or in JNK1-silenced ( b ) human erythroblasts by immunoblotting. c Phosphorylation of Tyr-15 of Cdk1 in P38α +/ − and P38α − / − erythroblasts. d – f Human erythroblasts transduced with control or cdk1-specific shRNA and treated with vehicle or SP600125. Protein levels of Ezh2 and Cdk1 by immunoblotting ( d ), mRNA expression of Bim by q-PCR ( e ), and representative flow cytometry profile (left) and quantification (right) of apoptotic human erythroblasts ( f ) ( n = 4). g Schematic outlining a proposed mechanism mediating rewired JNK activation due to lack of P38 via Map3k4 and their downstream targets regulating erythropoiesis. Blots are representative of three independent experiments. Data are shown as mean ± s.e.m. * P

    Article Snippet: Antibodies against P38α (9212,1:1000), phospho-P38 (9211, 1:1000), JNK1(3708, 1:1000), Total JNK (9252, 1:1000), phosphor-JNK (9251,1:1000), Ezh2 (5246, 1:3000), LRF (13097, 1:1000), CDK1(28439, 1:1000), phosphor-CDK1(9111, 1:1000), P53 (2524, 1:1000), Myc tag (2272, 1:1000), and phosphor-T-P (9391, 1:1000) are from Cell Signaling.

    Techniques: Inhibition, Transduction, shRNA, Expressing, Polymerase Chain Reaction, Flow Cytometry, Cytometry, Activation Assay

    Upregulated Map3k4 due to P38α deficiency mediates JNK activation in erythroblasts. a In sorted erythroblasts from P38α +/ − and P38α − / − mice, phosphorylated JNK (46 and 54 kDa) was detected by immunoblotting. b Gene set enrichment analysis (GSEA) of the P38 pathway based on gene expression profiling of sorted CD71 high Ter119 + erythroblasts from P38α +/ − and P38α − / − mice during recovery from PHZ challenge. NES normalized enrichment score; FDR false discovery rate q -value. c Heat map of P38α-related genes. d mRNA levels of key P38 and JNK regulatory genes in sorted P38α +/ − and P38α − / − CD71 high Ter119 + erythroblasts by q-PCR. e Western blot (left) and densitometry analysis (right) of Map3k4 protein level in P38α +/ − and P38α − / − erythroblasts. Data are shown as mean ± s.e.m from three separate experiments. f Sorted GFP + P38α +/ − and P38α − / − cells transduced with control or Map3k4-specific shRNA. Expression of phosphorylated JNK and Map3k4 as assessed by immunoblotting. g Expression of phosphorylated JNK and Map3k4 in human erythroblasts cultured with DMSO or SB203580. h Schematic diagram demonstrating mechanism by which loss of P38α rewires increased Map3k4 to exclusively activate JNK. Blots are representative of three independent experiments. Data are shown as mean ± s.e.m. * P

    Journal: Nature Communications

    Article Title: P38α/JNK signaling restrains erythropoiesis by suppressing Ezh2-mediated epigenetic silencing of Bim

    doi: 10.1038/s41467-018-05955-2

    Figure Lengend Snippet: Upregulated Map3k4 due to P38α deficiency mediates JNK activation in erythroblasts. a In sorted erythroblasts from P38α +/ − and P38α − / − mice, phosphorylated JNK (46 and 54 kDa) was detected by immunoblotting. b Gene set enrichment analysis (GSEA) of the P38 pathway based on gene expression profiling of sorted CD71 high Ter119 + erythroblasts from P38α +/ − and P38α − / − mice during recovery from PHZ challenge. NES normalized enrichment score; FDR false discovery rate q -value. c Heat map of P38α-related genes. d mRNA levels of key P38 and JNK regulatory genes in sorted P38α +/ − and P38α − / − CD71 high Ter119 + erythroblasts by q-PCR. e Western blot (left) and densitometry analysis (right) of Map3k4 protein level in P38α +/ − and P38α − / − erythroblasts. Data are shown as mean ± s.e.m from three separate experiments. f Sorted GFP + P38α +/ − and P38α − / − cells transduced with control or Map3k4-specific shRNA. Expression of phosphorylated JNK and Map3k4 as assessed by immunoblotting. g Expression of phosphorylated JNK and Map3k4 in human erythroblasts cultured with DMSO or SB203580. h Schematic diagram demonstrating mechanism by which loss of P38α rewires increased Map3k4 to exclusively activate JNK. Blots are representative of three independent experiments. Data are shown as mean ± s.e.m. * P

    Article Snippet: Antibodies against P38α (9212,1:1000), phospho-P38 (9211, 1:1000), JNK1(3708, 1:1000), Total JNK (9252, 1:1000), phosphor-JNK (9251,1:1000), Ezh2 (5246, 1:3000), LRF (13097, 1:1000), CDK1(28439, 1:1000), phosphor-CDK1(9111, 1:1000), P53 (2524, 1:1000), Myc tag (2272, 1:1000), and phosphor-T-P (9391, 1:1000) are from Cell Signaling.

    Techniques: Activation Assay, Mouse Assay, Expressing, Polymerase Chain Reaction, Western Blot, Transduction, shRNA, Cell Culture

    Bim but not P53 modulates erythroblast apoptosis downstream of JNK. a Human erythroblasts treated with DMSO or SP600125, P53 and Noxa expression measured by immunoblotting. b Time course of P53 protein expression in human erythroblasts treated with DMSO or SP600125 in the presence of cycloheximide (CHX). c P53 mRNA (left) and protein (right) level in P38α +/ − and P38α − / − erythroblasts measured by q-PCR or western blot. d GSEA analysis of P53 pathway in P38α +/ − and P38α − / − erythroblasts. e mRNA (left) and protein (right) level of Bim in human erythroblasts triggered by SP600125 at 12 h by q-PCR or immunoblotting. f Bim mRNA expression in human erythroblasts induced by JNK1 shRNA. g Representative flow plots (left) and Quantification (right) of SP600125-induced apoptosis of human erythroblasts transduced with control or Bim-specific shRNA ( n = 4). Blots are representative of three independent experiments. Data are shown as mean ± s.e.m. * P

    Journal: Nature Communications

    Article Title: P38α/JNK signaling restrains erythropoiesis by suppressing Ezh2-mediated epigenetic silencing of Bim

    doi: 10.1038/s41467-018-05955-2

    Figure Lengend Snippet: Bim but not P53 modulates erythroblast apoptosis downstream of JNK. a Human erythroblasts treated with DMSO or SP600125, P53 and Noxa expression measured by immunoblotting. b Time course of P53 protein expression in human erythroblasts treated with DMSO or SP600125 in the presence of cycloheximide (CHX). c P53 mRNA (left) and protein (right) level in P38α +/ − and P38α − / − erythroblasts measured by q-PCR or western blot. d GSEA analysis of P53 pathway in P38α +/ − and P38α − / − erythroblasts. e mRNA (left) and protein (right) level of Bim in human erythroblasts triggered by SP600125 at 12 h by q-PCR or immunoblotting. f Bim mRNA expression in human erythroblasts induced by JNK1 shRNA. g Representative flow plots (left) and Quantification (right) of SP600125-induced apoptosis of human erythroblasts transduced with control or Bim-specific shRNA ( n = 4). Blots are representative of three independent experiments. Data are shown as mean ± s.e.m. * P

    Article Snippet: Antibodies against P38α (9212,1:1000), phospho-P38 (9211, 1:1000), JNK1(3708, 1:1000), Total JNK (9252, 1:1000), phosphor-JNK (9251,1:1000), Ezh2 (5246, 1:3000), LRF (13097, 1:1000), CDK1(28439, 1:1000), phosphor-CDK1(9111, 1:1000), P53 (2524, 1:1000), Myc tag (2272, 1:1000), and phosphor-T-P (9391, 1:1000) are from Cell Signaling.

    Techniques: Expressing, Polymerase Chain Reaction, Western Blot, shRNA, Flow Cytometry, Transduction

    JNK promotes survival of erythroblasts independent of EPO. a Phosphorylation of JNK in starved human erythroblasts stimulated with EPO (2 U ml −1 ) at indicated time points. b Representative flow cytometry profile (left) and quantification (right) of apoptotic human erythroblasts induced by SP600125 (20 µM) for 36 h ( n = 5). c Representative flow cytometry plots (left) and quantification (right) of apoptotic human erythroblasts by JNK1-specific shRNA ( n = 4). d HCT and RBC counts on day 0 ( n = 8) and on indicated time points ( n = 5) in P38α +/ − and P38α − / − mice treated with vehicle or SP600125. e Number ( n = 4) and apoptosis ( n = 5) of erythroblast subsets at fourth week in P38α +/ − and P38α − / − mice exposed to vehicle or SP600125. f , g mRNA ( f ) and protein ( g ) expression of JunD from sorted P38α +/ − and P38α − / − erythroblasts during recovery (day 4) from PHZ-induced anemia. h JunD protein levels in SP600125 (20 µM) treated human erythroblasts by immunoblotting. i Representative flow cytometry profile (left) and quantification (right) of apoptotic human erythroblasts induced by JunD-specific shRNA ( n = 4). Blots are representative of three independent experiments. Data are shown as mean ± s.e.m. * P

    Journal: Nature Communications

    Article Title: P38α/JNK signaling restrains erythropoiesis by suppressing Ezh2-mediated epigenetic silencing of Bim

    doi: 10.1038/s41467-018-05955-2

    Figure Lengend Snippet: JNK promotes survival of erythroblasts independent of EPO. a Phosphorylation of JNK in starved human erythroblasts stimulated with EPO (2 U ml −1 ) at indicated time points. b Representative flow cytometry profile (left) and quantification (right) of apoptotic human erythroblasts induced by SP600125 (20 µM) for 36 h ( n = 5). c Representative flow cytometry plots (left) and quantification (right) of apoptotic human erythroblasts by JNK1-specific shRNA ( n = 4). d HCT and RBC counts on day 0 ( n = 8) and on indicated time points ( n = 5) in P38α +/ − and P38α − / − mice treated with vehicle or SP600125. e Number ( n = 4) and apoptosis ( n = 5) of erythroblast subsets at fourth week in P38α +/ − and P38α − / − mice exposed to vehicle or SP600125. f , g mRNA ( f ) and protein ( g ) expression of JunD from sorted P38α +/ − and P38α − / − erythroblasts during recovery (day 4) from PHZ-induced anemia. h JunD protein levels in SP600125 (20 µM) treated human erythroblasts by immunoblotting. i Representative flow cytometry profile (left) and quantification (right) of apoptotic human erythroblasts induced by JunD-specific shRNA ( n = 4). Blots are representative of three independent experiments. Data are shown as mean ± s.e.m. * P

    Article Snippet: Antibodies against P38α (9212,1:1000), phospho-P38 (9211, 1:1000), JNK1(3708, 1:1000), Total JNK (9252, 1:1000), phosphor-JNK (9251,1:1000), Ezh2 (5246, 1:3000), LRF (13097, 1:1000), CDK1(28439, 1:1000), phosphor-CDK1(9111, 1:1000), P53 (2524, 1:1000), Myc tag (2272, 1:1000), and phosphor-T-P (9391, 1:1000) are from Cell Signaling.

    Techniques: Flow Cytometry, Cytometry, shRNA, Mouse Assay, Expressing

    Ber and Y suppress IκBα, ERK, JNK and IRF3 phosphorylation as well as NF-κB activation, but not p38 MAPK phosphorylation in LPS-treated macrophages. Mouse peritoneal macrophages were incubated with vehicle, Ber (2.0 µM), Ber (2.0 µM)+Y (5.0 µM) or Y (5.0 µM) for 2 h and then treated with LPS (100 ng/ml) for another 1 h. Whole cell lysates were examined for the presence of total and phosphorylated form of IκBα, ERK, JNK, p38 MAPK and IRF3 using Western blotting. Data in graph are presented as the mean of the ratio of phosphorylated protein to total protein. Nuclear extracts were also used to measure the NF-κB activity using NF-κB p65 transcription factor assay kit, and NF-κB activity was normalized to control macrophages. n = 3−4. *P

    Journal: PLoS ONE

    Article Title: Yohimbine Enhances Protection of Berberine against LPS-Induced Mouse Lethality through Multiple Mechanisms

    doi: 10.1371/journal.pone.0052863

    Figure Lengend Snippet: Ber and Y suppress IκBα, ERK, JNK and IRF3 phosphorylation as well as NF-κB activation, but not p38 MAPK phosphorylation in LPS-treated macrophages. Mouse peritoneal macrophages were incubated with vehicle, Ber (2.0 µM), Ber (2.0 µM)+Y (5.0 µM) or Y (5.0 µM) for 2 h and then treated with LPS (100 ng/ml) for another 1 h. Whole cell lysates were examined for the presence of total and phosphorylated form of IκBα, ERK, JNK, p38 MAPK and IRF3 using Western blotting. Data in graph are presented as the mean of the ratio of phosphorylated protein to total protein. Nuclear extracts were also used to measure the NF-κB activity using NF-κB p65 transcription factor assay kit, and NF-κB activity was normalized to control macrophages. n = 3−4. *P

    Article Snippet: Antibodies, including IκBα, ERK, JNK, p38MAPK, NF-κB, IRF3, Tyk2, STAT1, phosphorylated IκBα (p-IκBα), phosphorylated JNK (p-JNK), phosphorylated ERK(p-ERK), phosphorylated p38MAPK (p-p38 MAPK), phosphorylated IRF3(p-IRF3), phosphorylated Tyk2 (p-Tyk2), phosphorylated STAT1(p-STAT1) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and horseradish peroxidase -labeled secondary antibody were from Cell Signaling Technology, Inc. (Danvers, MA, USA).

    Techniques: Activation Assay, Incubation, Western Blot, Activity Assay, Transcription Factor Assay

    Effects of Ber or/and Y on IκBα, ERK, JNK and p38 MAPK phosphorylation as well as NF-κB activation in spleen of LPS-challenged mice. Spleen was removed 1 h after LPS or normal saline injection, extraction of total protein was analyzed for the presence of total and phosphorylated forms of IκBα, ERK, JNK and p38 MAPK using Western blot, cytoplasmic extracts were subjected to Western blot analysis for determining NF-κB p65 level, nuclear extracts were used to measure the NF-κB activity using NF-κB p65 transcription factor assay kit, and NF-κB activity was normalized to control. n = 5−6 . * P

    Journal: PLoS ONE

    Article Title: Yohimbine Enhances Protection of Berberine against LPS-Induced Mouse Lethality through Multiple Mechanisms

    doi: 10.1371/journal.pone.0052863

    Figure Lengend Snippet: Effects of Ber or/and Y on IκBα, ERK, JNK and p38 MAPK phosphorylation as well as NF-κB activation in spleen of LPS-challenged mice. Spleen was removed 1 h after LPS or normal saline injection, extraction of total protein was analyzed for the presence of total and phosphorylated forms of IκBα, ERK, JNK and p38 MAPK using Western blot, cytoplasmic extracts were subjected to Western blot analysis for determining NF-κB p65 level, nuclear extracts were used to measure the NF-κB activity using NF-κB p65 transcription factor assay kit, and NF-κB activity was normalized to control. n = 5−6 . * P

    Article Snippet: Antibodies, including IκBα, ERK, JNK, p38MAPK, NF-κB, IRF3, Tyk2, STAT1, phosphorylated IκBα (p-IκBα), phosphorylated JNK (p-JNK), phosphorylated ERK(p-ERK), phosphorylated p38MAPK (p-p38 MAPK), phosphorylated IRF3(p-IRF3), phosphorylated Tyk2 (p-Tyk2), phosphorylated STAT1(p-STAT1) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and horseradish peroxidase -labeled secondary antibody were from Cell Signaling Technology, Inc. (Danvers, MA, USA).

    Techniques: Activation Assay, Mouse Assay, Injection, Western Blot, Activity Assay, Transcription Factor Assay

    Ran(K152A) enhances the transformed phenotype exhibited by SKBR3 breast cancer cells. A , A single SKBR3 cell clone stably expressing either the vector-alone, wild-type Ran, or Ran(K152A) were subjected to anchorage-independent growth (soft agar) assays. The experiments were performed three times and the resulting colonies that formed in each experiment were counted, averaged together, and graphed. The error bars represent standard deviation. Representative images of the soft agar assay are shown. B , SKBR3 cells stably expressing the vector-alone, wild-type Ran, or Ran(K152A) were serum-starved overnight and lysed. As a positive control, an additional plate of serum-starved SKBR3 cells expressing the vector alone was stimulated with medium containing serum for 10 min prior to being lysed. The cell extracts were analyzed by Western blot analysis using phospho-ERK, phospho-JNK, and HA antibodies. Similar results were obtained using additional clones (data not shown).

    Journal: The Journal of Biological Chemistry

    Article Title:

    doi: 10.1074/jbc.M111.306514

    Figure Lengend Snippet: Ran(K152A) enhances the transformed phenotype exhibited by SKBR3 breast cancer cells. A , A single SKBR3 cell clone stably expressing either the vector-alone, wild-type Ran, or Ran(K152A) were subjected to anchorage-independent growth (soft agar) assays. The experiments were performed three times and the resulting colonies that formed in each experiment were counted, averaged together, and graphed. The error bars represent standard deviation. Representative images of the soft agar assay are shown. B , SKBR3 cells stably expressing the vector-alone, wild-type Ran, or Ran(K152A) were serum-starved overnight and lysed. As a positive control, an additional plate of serum-starved SKBR3 cells expressing the vector alone was stimulated with medium containing serum for 10 min prior to being lysed. The cell extracts were analyzed by Western blot analysis using phospho-ERK, phospho-JNK, and HA antibodies. Similar results were obtained using additional clones (data not shown).

    Article Snippet: The HA antibody was obtained from Covance, whereas the phospho-ERK and phospho-JNK antibodies were from Cell Signaling.

    Techniques: Transformation Assay, Stable Transfection, Expressing, Plasmid Preparation, Standard Deviation, Soft Agar Assay, Positive Control, Western Blot, Clone Assay

    SMOC-2 is important for Ran promoted cellular transformation in human mammary SKBR3 adenocarcinoma cells. A , SKBR3 cells stably expressing the vector alone, Ran(K152A), or Ran(K152A) and SMOC-2 siRNAs (denoted as Kd 1 , 2 , or 3 ) were subjected to anchorage-independent growth (soft agar) assays. The experiments were performed three times, and the resulting colonies that formed in each experiment were counted, averaged, and graphed. The error bars represent S.D. B , SKBR3 cells stably expressing the vector alone, Ran(K152A), Ran(K152A), and SMOC-2 siRNA, or Ran(K152A), SMOC-2 siRNA, and an siRNA-insensitive form of SMOC-2 (SMOC-2 mutant) were subjected to anchorage-independent growth (soft agar) assays. The experiments were performed three times, and the resulting colonies that formed in each experiment were counted, and the data were averaged and graphed as a percent of the control Ran(K152A). The error bars represent S.D. C and D , serum-starved SKBR3 cells stably expressing the vector alone, Ran(K152A), or Ran(K152A) and SMOC-2 siRNAs were lysed. As a positive control, an additional plate of serum-starved SKBR3 cells overexpressing the vector alone was stimulated with medium containing serum for 10 min prior to being lysed. The cell extracts were analyzed by Western blot analysis using phospho-ERK, phospho-JNK, HA, and vinculin antibodies.

    Journal: The Journal of Biological Chemistry

    Article Title:

    doi: 10.1074/jbc.M111.306514

    Figure Lengend Snippet: SMOC-2 is important for Ran promoted cellular transformation in human mammary SKBR3 adenocarcinoma cells. A , SKBR3 cells stably expressing the vector alone, Ran(K152A), or Ran(K152A) and SMOC-2 siRNAs (denoted as Kd 1 , 2 , or 3 ) were subjected to anchorage-independent growth (soft agar) assays. The experiments were performed three times, and the resulting colonies that formed in each experiment were counted, averaged, and graphed. The error bars represent S.D. B , SKBR3 cells stably expressing the vector alone, Ran(K152A), Ran(K152A), and SMOC-2 siRNA, or Ran(K152A), SMOC-2 siRNA, and an siRNA-insensitive form of SMOC-2 (SMOC-2 mutant) were subjected to anchorage-independent growth (soft agar) assays. The experiments were performed three times, and the resulting colonies that formed in each experiment were counted, and the data were averaged and graphed as a percent of the control Ran(K152A). The error bars represent S.D. C and D , serum-starved SKBR3 cells stably expressing the vector alone, Ran(K152A), or Ran(K152A) and SMOC-2 siRNAs were lysed. As a positive control, an additional plate of serum-starved SKBR3 cells overexpressing the vector alone was stimulated with medium containing serum for 10 min prior to being lysed. The cell extracts were analyzed by Western blot analysis using phospho-ERK, phospho-JNK, HA, and vinculin antibodies.

    Article Snippet: The HA antibody was obtained from Covance, whereas the phospho-ERK and phospho-JNK antibodies were from Cell Signaling.

    Techniques: Transformation Assay, Stable Transfection, Expressing, Plasmid Preparation, Mutagenesis, Positive Control, Western Blot

    Activated Ran mutants induce cell transformation. A , NIH-3T3 cells transiently expressing the vector alone, wild-type Ran, the activated Ran mutants, or the constitutively active Ras(G12V) mutant were subjected to anchorage-independent growth (soft agar) assays. The experiments were performed three times, and the resulting colonies that formed in each experiment were counted, averaged together, and graphed. Representative images of the soft agar assay are shown. B , NIH-3T3 cells transiently expressing the vector alone, wild-type Ran, or Ran(K152A) were serum-starved overnight and lysed. As a positive control, an additional plate of serum-starved NIH-3T3 cells expressing the vector alone was stimulated with medium containing serum for 10 min prior to being lysed. The cell extracts were then subjected to Western blot analysis using phospho-ERK, phospho-JNK, HA, and vinculin antibodies. C , NIH-3T3 cells expressing the vector-alone or stably expressing Ran(K152A) were subjected to cell invasion assays under serum-starved and serum-stimulated conditions. The cells that invaded for each condition were counted and graphed. The results shown represent the average of three separate experiments. The error bars in A and C represent S.D.

    Journal: The Journal of Biological Chemistry

    Article Title:

    doi: 10.1074/jbc.M111.306514

    Figure Lengend Snippet: Activated Ran mutants induce cell transformation. A , NIH-3T3 cells transiently expressing the vector alone, wild-type Ran, the activated Ran mutants, or the constitutively active Ras(G12V) mutant were subjected to anchorage-independent growth (soft agar) assays. The experiments were performed three times, and the resulting colonies that formed in each experiment were counted, averaged together, and graphed. Representative images of the soft agar assay are shown. B , NIH-3T3 cells transiently expressing the vector alone, wild-type Ran, or Ran(K152A) were serum-starved overnight and lysed. As a positive control, an additional plate of serum-starved NIH-3T3 cells expressing the vector alone was stimulated with medium containing serum for 10 min prior to being lysed. The cell extracts were then subjected to Western blot analysis using phospho-ERK, phospho-JNK, HA, and vinculin antibodies. C , NIH-3T3 cells expressing the vector-alone or stably expressing Ran(K152A) were subjected to cell invasion assays under serum-starved and serum-stimulated conditions. The cells that invaded for each condition were counted and graphed. The results shown represent the average of three separate experiments. The error bars in A and C represent S.D.

    Article Snippet: The HA antibody was obtained from Covance, whereas the phospho-ERK and phospho-JNK antibodies were from Cell Signaling.

    Techniques: Transformation Assay, Expressing, Plasmid Preparation, Mutagenesis, Soft Agar Assay, Positive Control, Western Blot, Stable Transfection

    Light exposure increased the expression of p-MAPKs in the retina. MAPKs activation in retina, represented by the levels of p-ERK1/2, p-JNK and p-P38, were detected with immunofluorescence. The green arrows points to the ONL, and the yellow arrows points to the MAPKs enhancement area after light exposure. p-ERK1/2 (a), p-JNK (b) and p-P38 (c) were elevated in the rat model of light-induced retinal degeneration, mainly in the ONL, on days 3 and 5 when compared with their levels in the normal retina. MAPK: Mitogen-activated protein kinase; p-ERK1/2: Phosphorylated-extracellular regulated kinase 1/2; JNK: c-Jun N-terminal kinase; ONL: Outer nuclear layer; INL: Inner nuclear layer; GCL: Ganglion cell layer.

    Journal: Chinese Medical Journal

    Article Title: Effect of Phosphorylated-Extracellular Regulated Kinase 1/2 Inhibitor on Retina from Light-induced Photoreceptor Degeneration

    doi: 10.4103/0366-6999.246064

    Figure Lengend Snippet: Light exposure increased the expression of p-MAPKs in the retina. MAPKs activation in retina, represented by the levels of p-ERK1/2, p-JNK and p-P38, were detected with immunofluorescence. The green arrows points to the ONL, and the yellow arrows points to the MAPKs enhancement area after light exposure. p-ERK1/2 (a), p-JNK (b) and p-P38 (c) were elevated in the rat model of light-induced retinal degeneration, mainly in the ONL, on days 3 and 5 when compared with their levels in the normal retina. MAPK: Mitogen-activated protein kinase; p-ERK1/2: Phosphorylated-extracellular regulated kinase 1/2; JNK: c-Jun N-terminal kinase; ONL: Outer nuclear layer; INL: Inner nuclear layer; GCL: Ganglion cell layer.

    Article Snippet: Antibodies directed against the following proteins were used: p-P38 (1:100, Cat #4511, CST, Boston, MA, USA), p-JNK (1:100, Cat #4668, CST, Boston, MA, USA), or p-ERK1/2 (1:100, Cat #4370, CST, Boston, MA, USA).

    Techniques: Expressing, Activation Assay, Immunofluorescence

    Rosiglitazone counteracts JNK-induced β-cell dysfunction. GTT ( A ) and the corresponding area under the curve (AUC) for the first 60 min ( B ), glucose-stimulated insulin secretion ( C ), the corresponding AUC for the first 15 min ( D ), and ITT ( E ) performed in control and MKK7D 2-month-old mice treated for 10 days with rosiglitazone (Rosi) or PBS as indicated. F : Insulin secretion of pancreatic islets isolated from control and MKK7D mice treated with PBS or rosiglitazone. Islets were stimulated with glucose and insulin as indicated. G : Immunoblot analysis performed in extracts of pancreatic islets from control and MKK7D mice treated with rosiglitazone and insulin (Ins) (20 min) as indicated. The ratios of phospho-Akt (P-Akt) and phospho-JNK (P-JNK) to AKT and JNK, respectively, are represented in the right panel . Assays were performed with at least 10 animals per group and repeated at least 3 times. * P

    Journal: Diabetes

    Article Title: In Vivo JNK Activation in Pancreatic ?-Cells Leads to Glucose Intolerance Caused by Insulin Resistance in Pancreas

    doi: 10.2337/db12-1097

    Figure Lengend Snippet: Rosiglitazone counteracts JNK-induced β-cell dysfunction. GTT ( A ) and the corresponding area under the curve (AUC) for the first 60 min ( B ), glucose-stimulated insulin secretion ( C ), the corresponding AUC for the first 15 min ( D ), and ITT ( E ) performed in control and MKK7D 2-month-old mice treated for 10 days with rosiglitazone (Rosi) or PBS as indicated. F : Insulin secretion of pancreatic islets isolated from control and MKK7D mice treated with PBS or rosiglitazone. Islets were stimulated with glucose and insulin as indicated. G : Immunoblot analysis performed in extracts of pancreatic islets from control and MKK7D mice treated with rosiglitazone and insulin (Ins) (20 min) as indicated. The ratios of phospho-Akt (P-Akt) and phospho-JNK (P-JNK) to AKT and JNK, respectively, are represented in the right panel . Assays were performed with at least 10 animals per group and repeated at least 3 times. * P

    Article Snippet: Dual specificity mitogen-activated protein kinase kinase 7 (MKK7), JNK, green fluorescent protein (GFP), Akt, phospho-Akt, caspase 3, phospho-JNK, Pdx1, and GLUT2 were detected using the antibodies sc-7104, sc-474 (Santa Cruz Biotechnology), 11814460001 (Roche), 9272S, 4060P, 9662 (Cell Signaling), 36-9300 (Invitrogen), 07-696 (Upstate), and AB1342 (Chemicon), respectively.

    Techniques: Mouse Assay, Isolation

    Tamoxifen-inducible JNK activation in adult mice causes glucose intolerance. A : GTT of control and MKK7DTam 8-week-old mice before treatment with tamoxifen. B : Immunoblot analysis of GFP, MKK7, phosphorylated JNK (P-JNK), and JNK in extracts from isolated pancreatic islets of tamoxifen-treated control and MKK7DTam mice. C , D , and E : GTT, ITT, and glucose-stimulated insulin secretion, respectively, in tamoxifen-treated control and MKK7DTam mice. F : Insulin secretion of pancreatic islets isolated from tamoxifen-treated control and MKK7DTam mice stimulated with glucose and insulin as indicated. Unless indicated, assays were performed 2 weeks after tamoxifen treatment with 10 animals per group. * P

    Journal: Diabetes

    Article Title: In Vivo JNK Activation in Pancreatic ?-Cells Leads to Glucose Intolerance Caused by Insulin Resistance in Pancreas

    doi: 10.2337/db12-1097

    Figure Lengend Snippet: Tamoxifen-inducible JNK activation in adult mice causes glucose intolerance. A : GTT of control and MKK7DTam 8-week-old mice before treatment with tamoxifen. B : Immunoblot analysis of GFP, MKK7, phosphorylated JNK (P-JNK), and JNK in extracts from isolated pancreatic islets of tamoxifen-treated control and MKK7DTam mice. C , D , and E : GTT, ITT, and glucose-stimulated insulin secretion, respectively, in tamoxifen-treated control and MKK7DTam mice. F : Insulin secretion of pancreatic islets isolated from tamoxifen-treated control and MKK7DTam mice stimulated with glucose and insulin as indicated. Unless indicated, assays were performed 2 weeks after tamoxifen treatment with 10 animals per group. * P

    Article Snippet: Dual specificity mitogen-activated protein kinase kinase 7 (MKK7), JNK, green fluorescent protein (GFP), Akt, phospho-Akt, caspase 3, phospho-JNK, Pdx1, and GLUT2 were detected using the antibodies sc-7104, sc-474 (Santa Cruz Biotechnology), 11814460001 (Roche), 9272S, 4060P, 9662 (Cell Signaling), 36-9300 (Invitrogen), 07-696 (Upstate), and AB1342 (Chemicon), respectively.

    Techniques: Activation Assay, Mouse Assay, Isolation

    JNK activation in pancreatic β-cells affects neither the morphology nor the insulin content of the islets. Pancreatic histological sections of control and MKK7D animals stained with hematoxylin-eosin ( A ) or analyzed by immunohistochemistry with antibodies against insulin and glucagon ( B ). Average β-cell volume to islet volume (Vv) ( C ) and islet area ( D ), insulin content of pancreas ( E ), pancreas weight relative to body weight ( F ), insulin content in isolated islets ( G ) from control and MKK7D mice. H : Immunoblot analysis of caspase 3 in extracts from isolated islets of control and MKK7D mice. Positive control was an extract from neurons treated overnight with staurosporine. I : Caspase 3 activity in extracts from isolated islets untreated or incubated for 20 h with 10 μmol/L staurosporine (STS) of control and MKK7D mice. Assays were performed in 3-month-old mice with at least 10 animals or 200 islets per group. ** P

    Journal: Diabetes

    Article Title: In Vivo JNK Activation in Pancreatic ?-Cells Leads to Glucose Intolerance Caused by Insulin Resistance in Pancreas

    doi: 10.2337/db12-1097

    Figure Lengend Snippet: JNK activation in pancreatic β-cells affects neither the morphology nor the insulin content of the islets. Pancreatic histological sections of control and MKK7D animals stained with hematoxylin-eosin ( A ) or analyzed by immunohistochemistry with antibodies against insulin and glucagon ( B ). Average β-cell volume to islet volume (Vv) ( C ) and islet area ( D ), insulin content of pancreas ( E ), pancreas weight relative to body weight ( F ), insulin content in isolated islets ( G ) from control and MKK7D mice. H : Immunoblot analysis of caspase 3 in extracts from isolated islets of control and MKK7D mice. Positive control was an extract from neurons treated overnight with staurosporine. I : Caspase 3 activity in extracts from isolated islets untreated or incubated for 20 h with 10 μmol/L staurosporine (STS) of control and MKK7D mice. Assays were performed in 3-month-old mice with at least 10 animals or 200 islets per group. ** P

    Article Snippet: Dual specificity mitogen-activated protein kinase kinase 7 (MKK7), JNK, green fluorescent protein (GFP), Akt, phospho-Akt, caspase 3, phospho-JNK, Pdx1, and GLUT2 were detected using the antibodies sc-7104, sc-474 (Santa Cruz Biotechnology), 11814460001 (Roche), 9272S, 4060P, 9662 (Cell Signaling), 36-9300 (Invitrogen), 07-696 (Upstate), and AB1342 (Chemicon), respectively.

    Techniques: Activation Assay, Staining, Immunohistochemistry, Isolation, Mouse Assay, Positive Control, Activity Assay, Incubation

    JNK activation in pancreatic β-cells leads to glucose intolerance as a result of an impaired capacity to increase insulinemia in response to hyperglycemia. A : Diagram of the GFP loxP -MKK7D transgene. Transgenic mice constitutively express GFP under the control of the CMV enhancer/chicken β-actin promoter (control mice). When crossed with RIP-Cre mice, the GFP cassette is floxed in pancreatic β-cells, leading to the constitutive expression of MKK7D in this cell type (MKK7D mice). E-GFP-N1 (enhanced green fluorescent protein) encodes a red-shifted variant of wild-type GFP. B : Immunoblot analysis of GFP, MKK7, phospho-JNK (P-JNK), and JNK in extracts from isolated pancreatic islets of control and MKK7D mice (first to fourth panel). Fifth panel shows JNK activity assessed by immunocomplex assay. C : Body weight comparison of control and MKK7D mice. Plasma glucose ( D ) and insulin ( E ) level in 6-h-fasted control and MKK7D mice. GTT ( F ), ITT ( G ), and glucose-stimulated insulin secretion ( H ) in control and MKK7D mice. Assays were performed in 3-month-old mice with at least 10 animals per group. * P

    Journal: Diabetes

    Article Title: In Vivo JNK Activation in Pancreatic ?-Cells Leads to Glucose Intolerance Caused by Insulin Resistance in Pancreas

    doi: 10.2337/db12-1097

    Figure Lengend Snippet: JNK activation in pancreatic β-cells leads to glucose intolerance as a result of an impaired capacity to increase insulinemia in response to hyperglycemia. A : Diagram of the GFP loxP -MKK7D transgene. Transgenic mice constitutively express GFP under the control of the CMV enhancer/chicken β-actin promoter (control mice). When crossed with RIP-Cre mice, the GFP cassette is floxed in pancreatic β-cells, leading to the constitutive expression of MKK7D in this cell type (MKK7D mice). E-GFP-N1 (enhanced green fluorescent protein) encodes a red-shifted variant of wild-type GFP. B : Immunoblot analysis of GFP, MKK7, phospho-JNK (P-JNK), and JNK in extracts from isolated pancreatic islets of control and MKK7D mice (first to fourth panel). Fifth panel shows JNK activity assessed by immunocomplex assay. C : Body weight comparison of control and MKK7D mice. Plasma glucose ( D ) and insulin ( E ) level in 6-h-fasted control and MKK7D mice. GTT ( F ), ITT ( G ), and glucose-stimulated insulin secretion ( H ) in control and MKK7D mice. Assays were performed in 3-month-old mice with at least 10 animals per group. * P

    Article Snippet: Dual specificity mitogen-activated protein kinase kinase 7 (MKK7), JNK, green fluorescent protein (GFP), Akt, phospho-Akt, caspase 3, phospho-JNK, Pdx1, and GLUT2 were detected using the antibodies sc-7104, sc-474 (Santa Cruz Biotechnology), 11814460001 (Roche), 9272S, 4060P, 9662 (Cell Signaling), 36-9300 (Invitrogen), 07-696 (Upstate), and AB1342 (Chemicon), respectively.

    Techniques: Activation Assay, Transgenic Assay, Mouse Assay, Expressing, Variant Assay, Isolation, Activity Assay

    JNK activation in pancreatic β-cells impairs glucose-stimulated insulin secretion. Insulin secretion of pancreatic islets isolated from control and MKK7D mice stimulated with the indicated concentrations of glucose ( A ) and in the presence or absence of JNKi-1 (JNKi) as indicated ( B ). Analysis of GLUT2 expression by immunoblot in extracts from pancreatic islets ( C ) and subcellular localization by immunohistochemistry in pancreatic sections ( D ) of control and MKK7D mice. E : Insulin secretion of islets isolated from control and MKK7D mice in response to 2.8 mmol/L (−) and 24 mmol/L glucose (Glu), succinate, KIC, tolbutamide (Tolb), KCl, and IBMX, as indicated. Assays were performed in 3-month-old mice and repeated at least 3 times. * P

    Journal: Diabetes

    Article Title: In Vivo JNK Activation in Pancreatic ?-Cells Leads to Glucose Intolerance Caused by Insulin Resistance in Pancreas

    doi: 10.2337/db12-1097

    Figure Lengend Snippet: JNK activation in pancreatic β-cells impairs glucose-stimulated insulin secretion. Insulin secretion of pancreatic islets isolated from control and MKK7D mice stimulated with the indicated concentrations of glucose ( A ) and in the presence or absence of JNKi-1 (JNKi) as indicated ( B ). Analysis of GLUT2 expression by immunoblot in extracts from pancreatic islets ( C ) and subcellular localization by immunohistochemistry in pancreatic sections ( D ) of control and MKK7D mice. E : Insulin secretion of islets isolated from control and MKK7D mice in response to 2.8 mmol/L (−) and 24 mmol/L glucose (Glu), succinate, KIC, tolbutamide (Tolb), KCl, and IBMX, as indicated. Assays were performed in 3-month-old mice and repeated at least 3 times. * P

    Article Snippet: Dual specificity mitogen-activated protein kinase kinase 7 (MKK7), JNK, green fluorescent protein (GFP), Akt, phospho-Akt, caspase 3, phospho-JNK, Pdx1, and GLUT2 were detected using the antibodies sc-7104, sc-474 (Santa Cruz Biotechnology), 11814460001 (Roche), 9272S, 4060P, 9662 (Cell Signaling), 36-9300 (Invitrogen), 07-696 (Upstate), and AB1342 (Chemicon), respectively.

    Techniques: Activation Assay, Isolation, Mouse Assay, Expressing, Immunohistochemistry

    JNK activation in pancreatic β-cells impairs insulin signaling. A : Insulin-induced insulin secretion of pancreatic islets isolated from control and MKK7D mice in the presence or absence of JNKi-1 (JNKi) as indicated. Secretions were performed in basal glucose concentration (2.8 mmol/L). B : Immunoblot analysis of phospho-Akt (P-Akt) and Akt in extracts of islets from control and MKK7D mice incubated with or without insulin (Ins), as indicated, for 20 min. The ratio of phospho-Akt to total Akt is represented in the bottom panel . C : Quantitative RT-PCR analysis of the indicated insulin target genes in islets from control and MKK7 mice incubated with or without insulin, as indicated, for 3 h. D : Immunoblot analysis of Pdx1 in extracts from pancreatic islets of control and MKK7D mice. The ratio of Pdx1 to loading control is represented in the bottom panel . E : Immunohistochemistry analysis of Pdx1 and insulin in pancreatic sections from control and MKK7D mice untreated (−) or treated with glucose (Glu) or insulin (Ins) for 30 min. Assays were performed in 3-month-old mice and repeated at least 3 times. * P

    Journal: Diabetes

    Article Title: In Vivo JNK Activation in Pancreatic ?-Cells Leads to Glucose Intolerance Caused by Insulin Resistance in Pancreas

    doi: 10.2337/db12-1097

    Figure Lengend Snippet: JNK activation in pancreatic β-cells impairs insulin signaling. A : Insulin-induced insulin secretion of pancreatic islets isolated from control and MKK7D mice in the presence or absence of JNKi-1 (JNKi) as indicated. Secretions were performed in basal glucose concentration (2.8 mmol/L). B : Immunoblot analysis of phospho-Akt (P-Akt) and Akt in extracts of islets from control and MKK7D mice incubated with or without insulin (Ins), as indicated, for 20 min. The ratio of phospho-Akt to total Akt is represented in the bottom panel . C : Quantitative RT-PCR analysis of the indicated insulin target genes in islets from control and MKK7 mice incubated with or without insulin, as indicated, for 3 h. D : Immunoblot analysis of Pdx1 in extracts from pancreatic islets of control and MKK7D mice. The ratio of Pdx1 to loading control is represented in the bottom panel . E : Immunohistochemistry analysis of Pdx1 and insulin in pancreatic sections from control and MKK7D mice untreated (−) or treated with glucose (Glu) or insulin (Ins) for 30 min. Assays were performed in 3-month-old mice and repeated at least 3 times. * P

    Article Snippet: Dual specificity mitogen-activated protein kinase kinase 7 (MKK7), JNK, green fluorescent protein (GFP), Akt, phospho-Akt, caspase 3, phospho-JNK, Pdx1, and GLUT2 were detected using the antibodies sc-7104, sc-474 (Santa Cruz Biotechnology), 11814460001 (Roche), 9272S, 4060P, 9662 (Cell Signaling), 36-9300 (Invitrogen), 07-696 (Upstate), and AB1342 (Chemicon), respectively.

    Techniques: Activation Assay, Isolation, Mouse Assay, Concentration Assay, Incubation, Quantitative RT-PCR, Immunohistochemistry

    Aβ 1–40 -induced NF-κB pathway activation. A , B , Cytoplasmic [(C)] and nuclear [(N)] samples of hippocampus ( A ) and prefrontal cortex ( B ) from Swiss mice were probed with a p65 NF-κB antibody at the time points indicated. Data indicate that Aβ 1–40 induced a translocation of p65 NF-κB from the cytoplasm to the nucleus. Pretreatment with the specific antibody against mouse TNF-α (AbTNF-α; 10 ηg, i.c.v., per mouse) prevented Aβ 1–40 induction of p65 NF-κB translocation in hippocampus and prefrontal cortex. Immunoblot for lamin A/C was used as a nuclear loading control. Treatment with the reverse peptide Aβ 40–1 (6 h) had no significant effect on the p65 NF-κB translocation. As a positive control, LPS (2.5 μg, i.c.v., 1 h) treatment resulted in p65 NF-κB migration into the nucleus. C , D , Aβ 1–40 -induced p65 NF-κB translocation was prevented by pretreatment with NF-κB inhibitor PDTC (100 mg/kg, i.p.), but not by pretreatment with JNK inhibitor SP600125 (25 mg/kg, i.p.), in hippocampus ( C ) and prefrontal cortex ( D ). These data indicate that JNK/c-Jun and NF-κB pathways are independently activated by Aβ 1–40 intracerebroventricular injection. N, Naïve, untreated mice; C, control.

    Journal: The Journal of Neuroscience

    Article Title: Connecting TNF-α Signaling Pathways to iNOS Expression in a Mouse Model of Alzheimer's Disease: Relevance for the Behavioral and Synaptic Deficits Induced by Amyloid β Protein

    doi: 10.1523/JNEUROSCI.5047-06.2007

    Figure Lengend Snippet: Aβ 1–40 -induced NF-κB pathway activation. A , B , Cytoplasmic [(C)] and nuclear [(N)] samples of hippocampus ( A ) and prefrontal cortex ( B ) from Swiss mice were probed with a p65 NF-κB antibody at the time points indicated. Data indicate that Aβ 1–40 induced a translocation of p65 NF-κB from the cytoplasm to the nucleus. Pretreatment with the specific antibody against mouse TNF-α (AbTNF-α; 10 ηg, i.c.v., per mouse) prevented Aβ 1–40 induction of p65 NF-κB translocation in hippocampus and prefrontal cortex. Immunoblot for lamin A/C was used as a nuclear loading control. Treatment with the reverse peptide Aβ 40–1 (6 h) had no significant effect on the p65 NF-κB translocation. As a positive control, LPS (2.5 μg, i.c.v., 1 h) treatment resulted in p65 NF-κB migration into the nucleus. C , D , Aβ 1–40 -induced p65 NF-κB translocation was prevented by pretreatment with NF-κB inhibitor PDTC (100 mg/kg, i.p.), but not by pretreatment with JNK inhibitor SP600125 (25 mg/kg, i.p.), in hippocampus ( C ) and prefrontal cortex ( D ). These data indicate that JNK/c-Jun and NF-κB pathways are independently activated by Aβ 1–40 intracerebroventricular injection. N, Naïve, untreated mice; C, control.

    Article Snippet: The membranes were saturated by incubation with 10% nonfat dry milk solution and incubated overnight with one of the following antibodies: p65 NF-κB (sc-372, 1:1000), c- jun (sc-45, 1:1000), iNOS (sc-7271, 1:200), α-actin (sc-1615, 1:2000), JNK1 (sc-571, 1:1000), phosphorylated JNK (sc-6254, 1:1000) (all from Santa Cruz Biotechnology, Santa Cruz, CA), or lamin A/C (catalog #2032; Cell Signaling Technology).

    Techniques: Activation Assay, Mouse Assay, Translocation Assay, Positive Control, Migration, Injection

    Aβ 1–40 -induced expression of iNOS is dependent on JNK/c-Jun and NF-κB activation. Involvement of JNK/c-Jun and NF-κB signaling pathways on iNOS expression was verified in the hippocampus ( A ) and prefrontal cortex ( B ) 24 h after Aβ 1–40 intracerebroventricular administration. Top, Pretreatment with the selective inhibitor of JNK, SP600126 (50 mg/kg, i.p.), or NF-κB blocker PDTC (100 mg/kg, i.p.) 1 h before Aβ 1–40 treatment reduced iNOS expression in the hippocampus ( F (3,8) = 40.00; p

    Journal: The Journal of Neuroscience

    Article Title: Connecting TNF-α Signaling Pathways to iNOS Expression in a Mouse Model of Alzheimer's Disease: Relevance for the Behavioral and Synaptic Deficits Induced by Amyloid β Protein

    doi: 10.1523/JNEUROSCI.5047-06.2007

    Figure Lengend Snippet: Aβ 1–40 -induced expression of iNOS is dependent on JNK/c-Jun and NF-κB activation. Involvement of JNK/c-Jun and NF-κB signaling pathways on iNOS expression was verified in the hippocampus ( A ) and prefrontal cortex ( B ) 24 h after Aβ 1–40 intracerebroventricular administration. Top, Pretreatment with the selective inhibitor of JNK, SP600126 (50 mg/kg, i.p.), or NF-κB blocker PDTC (100 mg/kg, i.p.) 1 h before Aβ 1–40 treatment reduced iNOS expression in the hippocampus ( F (3,8) = 40.00; p

    Article Snippet: The membranes were saturated by incubation with 10% nonfat dry milk solution and incubated overnight with one of the following antibodies: p65 NF-κB (sc-372, 1:1000), c- jun (sc-45, 1:1000), iNOS (sc-7271, 1:200), α-actin (sc-1615, 1:2000), JNK1 (sc-571, 1:1000), phosphorylated JNK (sc-6254, 1:1000) (all from Santa Cruz Biotechnology, Santa Cruz, CA), or lamin A/C (catalog #2032; Cell Signaling Technology).

    Techniques: Expressing, Activation Assay

    Aβ 1–40 -induced JNK/c-Jun pathway activation. A , B , Activation of cytoplasmic JNK in response to aggregate Aβ 1–40 intracerebroventricular injection was detected in the hippocampus ( A ) and prefrontal cortex ( B ) of Swiss mice, at the time points indicated, by phosphospecific antibody for diphosphorylated JNK (p-JNK; at Thr 183 and Tyr 185 ). Blots were reprobed with anti-JNK to verify equal loading. C , D , Kinetics of c-Jun protein translocation from cytoplasm into the nucleus in the hippocampus ( C ) and prefrontal cortex ( D ) after Aβ 1–40 administration. Activation of the JNK pathway is dependent on TNF-α. Pretreatment with the specific antibody against mouse TNF-α (AbTNF-α; 10 ηg, i.c.v., per mouse) prevented Aβ 1–40 -induced JNK activation and subsequent c-Jun translocation in the hippocampus and prefrontal cortex. Immunoblot for lamin A/C was used as a nuclear loading control. N, Naïve, untreated mice. Treatment with the reverse peptide Aβ 40–1 (6 h) had no significant effect on the JNK/c-Jun activation. As a positive control, LPS (2.5 μg, i.c.v., 1 h) treatment induced activation in the JNK/c-Jun pathway. E , F , Pretreatment with JNK inhibitor SP600125 (25 mg/kg, i.p., 1 h before Aβ), but not with NF-κB inhibitor PDTC (100 mg/kg, i.p., 1 h before Aβ), prevented Aβ 1–40 -induced c-Jun translocation in the hippocampal ( E ) and cortical ( F ) homogenates. Brain samples were collected 6 h after Aβ 1–40 intracerebroventricular injection. These data indicate an association between JNK and c-Jun proteins.( C ), Cytoplasmatic; (N), nuclear.

    Journal: The Journal of Neuroscience

    Article Title: Connecting TNF-α Signaling Pathways to iNOS Expression in a Mouse Model of Alzheimer's Disease: Relevance for the Behavioral and Synaptic Deficits Induced by Amyloid β Protein

    doi: 10.1523/JNEUROSCI.5047-06.2007

    Figure Lengend Snippet: Aβ 1–40 -induced JNK/c-Jun pathway activation. A , B , Activation of cytoplasmic JNK in response to aggregate Aβ 1–40 intracerebroventricular injection was detected in the hippocampus ( A ) and prefrontal cortex ( B ) of Swiss mice, at the time points indicated, by phosphospecific antibody for diphosphorylated JNK (p-JNK; at Thr 183 and Tyr 185 ). Blots were reprobed with anti-JNK to verify equal loading. C , D , Kinetics of c-Jun protein translocation from cytoplasm into the nucleus in the hippocampus ( C ) and prefrontal cortex ( D ) after Aβ 1–40 administration. Activation of the JNK pathway is dependent on TNF-α. Pretreatment with the specific antibody against mouse TNF-α (AbTNF-α; 10 ηg, i.c.v., per mouse) prevented Aβ 1–40 -induced JNK activation and subsequent c-Jun translocation in the hippocampus and prefrontal cortex. Immunoblot for lamin A/C was used as a nuclear loading control. N, Naïve, untreated mice. Treatment with the reverse peptide Aβ 40–1 (6 h) had no significant effect on the JNK/c-Jun activation. As a positive control, LPS (2.5 μg, i.c.v., 1 h) treatment induced activation in the JNK/c-Jun pathway. E , F , Pretreatment with JNK inhibitor SP600125 (25 mg/kg, i.p., 1 h before Aβ), but not with NF-κB inhibitor PDTC (100 mg/kg, i.p., 1 h before Aβ), prevented Aβ 1–40 -induced c-Jun translocation in the hippocampal ( E ) and cortical ( F ) homogenates. Brain samples were collected 6 h after Aβ 1–40 intracerebroventricular injection. These data indicate an association between JNK and c-Jun proteins.( C ), Cytoplasmatic; (N), nuclear.

    Article Snippet: The membranes were saturated by incubation with 10% nonfat dry milk solution and incubated overnight with one of the following antibodies: p65 NF-κB (sc-372, 1:1000), c- jun (sc-45, 1:1000), iNOS (sc-7271, 1:200), α-actin (sc-1615, 1:2000), JNK1 (sc-571, 1:1000), phosphorylated JNK (sc-6254, 1:1000) (all from Santa Cruz Biotechnology, Santa Cruz, CA), or lamin A/C (catalog #2032; Cell Signaling Technology).

    Techniques: Activation Assay, Injection, Mouse Assay, Translocation Assay, Positive Control

    TBMS1 inhibits the phosphorylation of p38 and ERK1/2 in LPS-exposed BV-2 cells. The BV-2 cells were pretreated for 1 h with TBMS1 (1, 2 and 4 μM), then incubated with LPS (1 μg/mL) for 1 h. ( A ) The protein levels of p38, ERK1/2, JNK1/2 and their phosphorylated forms were tested by western blotting. The phosphorylation of p38 ( B ), ERK1/2 ( C ) and JNK1/2 ( D ) was analyzed relative to β-actin. The results are shown as mean ± SD of three independent experiments. # p

    Journal: International Journal of Molecular Sciences

    Article Title: Tubeimoside I Protects Dopaminergic Neurons Against Inflammation-Mediated Damage in Lipopolysaccharide (LPS)-Evoked Model of Parkinson’s Disease in Rats

    doi: 10.3390/ijms19082242

    Figure Lengend Snippet: TBMS1 inhibits the phosphorylation of p38 and ERK1/2 in LPS-exposed BV-2 cells. The BV-2 cells were pretreated for 1 h with TBMS1 (1, 2 and 4 μM), then incubated with LPS (1 μg/mL) for 1 h. ( A ) The protein levels of p38, ERK1/2, JNK1/2 and their phosphorylated forms were tested by western blotting. The phosphorylation of p38 ( B ), ERK1/2 ( C ) and JNK1/2 ( D ) was analyzed relative to β-actin. The results are shown as mean ± SD of three independent experiments. # p

    Article Snippet: After blocking with 5% skim milk, the polyvinylidene difluoride membranes were incubated with primary antibodies against TH (1:1000) (Abcam, Cambridge, UK), OX-42 (1:1000) (Abcam), COX-2 (1:2000) (Abcam), iNOS (1:2000) (Abcam), phospho-AKT (1:2000) (Cell Signaling Technology, MA, USA), phospho-JNK1/2 (1:2000) (Cell Signaling Technology), phospho-ERK1/2 (1:2000) (Cell Signaling Technology), phospho-p38 (1:2,000), phospho-NF-κB p65 (1:2000), JNK1/2 (1:2000), ERK1/2 (1:22,000) (Cell Signaling Technology), p38 (1:2000) (Cell Signaling Technology), AKT (1:2000) (Cell Signaling Technology), NF-κB p65 (1:2000) (Cell Signaling Technology) and β-actin (1:10,000) (Santa Cruz, CA, USA).

    Techniques: Incubation, Western Blot

    Resveratrol inhibits S. aureus -induced VCAM-1 expression and inflammatory signaling pathways. ( A ) Cells were pretreated with resveratrol for 24 h, and then incubated with S. aureus for 4 h or 6 h. The mRNA levels and promoter activity of VCAM-1 were determined. ( B ) Cells were pretreated with resveratrol for 24 h, and then incubated with S. aureus for 24 h. The THP-1 cells adherence was measured. ( C – E ) Cells were pretreated without or with resveratrol for 24 h, and then incubated with S. aureus for the indicated times. The expression of phospho-c-Src, phospho-PDGFR, phospho-p38 MAPK, phospho-JNK1/2, phospho-c-Jun, and phospho-ATF2 were determined by Western blot. ( F ) Cells were pretreated with resveratrol for 24 h, and then incubated with S. aureus for 4 h. ATF2 and c-Jun binding activities were analyzed by a ChIP assay. n = 3–4, # p

    Journal: International Journal of Molecular Sciences

    Article Title: Resveratrol Attenuates Staphylococcus Aureus-Induced Monocyte Adhesion through Downregulating PDGFR/AP-1 Activation in Human Lung Epithelial Cells

    doi: 10.3390/ijms19103058

    Figure Lengend Snippet: Resveratrol inhibits S. aureus -induced VCAM-1 expression and inflammatory signaling pathways. ( A ) Cells were pretreated with resveratrol for 24 h, and then incubated with S. aureus for 4 h or 6 h. The mRNA levels and promoter activity of VCAM-1 were determined. ( B ) Cells were pretreated with resveratrol for 24 h, and then incubated with S. aureus for 24 h. The THP-1 cells adherence was measured. ( C – E ) Cells were pretreated without or with resveratrol for 24 h, and then incubated with S. aureus for the indicated times. The expression of phospho-c-Src, phospho-PDGFR, phospho-p38 MAPK, phospho-JNK1/2, phospho-c-Jun, and phospho-ATF2 were determined by Western blot. ( F ) Cells were pretreated with resveratrol for 24 h, and then incubated with S. aureus for 4 h. ATF2 and c-Jun binding activities were analyzed by a ChIP assay. n = 3–4, # p

    Article Snippet: Anti-phospho-p38 MAPK, anti-phospho-JNK1/2, anti-phospho-p42/p44 MAPK, anti-phospho-c-Src, anti-phospho-PDGFR, anti-phospho-ATF2, and anti-phospho-c-Jun antibodies were obtained from Cell Signaling (Danver, MA, USA).

    Techniques: Expressing, Incubation, Activity Assay, Western Blot, Binding Assay, Chromatin Immunoprecipitation

    S. aureus induces AP-1 activation via c-Src/PDGFR/JNK1/2 and p38 MAPK pathways. Cells were pretreated without or with PP1, AG1296, SP600125, or SB202190 for 1 h, and then incubated with S. aureus for the indicated times. The expression of ( A ) phospho-c-Jun and ( B ) phospho-ATF2 was determined by Western blot. n = 3–4, # p

    Journal: International Journal of Molecular Sciences

    Article Title: Resveratrol Attenuates Staphylococcus Aureus-Induced Monocyte Adhesion through Downregulating PDGFR/AP-1 Activation in Human Lung Epithelial Cells

    doi: 10.3390/ijms19103058

    Figure Lengend Snippet: S. aureus induces AP-1 activation via c-Src/PDGFR/JNK1/2 and p38 MAPK pathways. Cells were pretreated without or with PP1, AG1296, SP600125, or SB202190 for 1 h, and then incubated with S. aureus for the indicated times. The expression of ( A ) phospho-c-Jun and ( B ) phospho-ATF2 was determined by Western blot. n = 3–4, # p

    Article Snippet: Anti-phospho-p38 MAPK, anti-phospho-JNK1/2, anti-phospho-p42/p44 MAPK, anti-phospho-c-Src, anti-phospho-PDGFR, anti-phospho-ATF2, and anti-phospho-c-Jun antibodies were obtained from Cell Signaling (Danver, MA, USA).

    Techniques: Activation Assay, Incubation, Expressing, Western Blot

    Schematic diagram illustrating the proposed signaling pathway involved in S. aureus -induced VCAM-1 expression in HPAEpiCs. S. aureus induces JNK1/2 and p38 MAPK activation via a c-Src/PDGFR pathway, which in turn initiates the activation of AP-1. Activated AP-1 is recruited to the promoter regions of VCAM-1, leading to an increase of VCAM-1 promoter activity and the expression of VCAM-1 mRNA and protein in HPAEpiCs. Moreover, resveratrol can reduce lung inflammation via the inhibition of VCAM-1 expression, monocyte adhesion, and the activation of c-Src, PDGFR, JNK1/2, p38 MAPK, and AP-1 induced by S. aureus .

    Journal: International Journal of Molecular Sciences

    Article Title: Resveratrol Attenuates Staphylococcus Aureus-Induced Monocyte Adhesion through Downregulating PDGFR/AP-1 Activation in Human Lung Epithelial Cells

    doi: 10.3390/ijms19103058

    Figure Lengend Snippet: Schematic diagram illustrating the proposed signaling pathway involved in S. aureus -induced VCAM-1 expression in HPAEpiCs. S. aureus induces JNK1/2 and p38 MAPK activation via a c-Src/PDGFR pathway, which in turn initiates the activation of AP-1. Activated AP-1 is recruited to the promoter regions of VCAM-1, leading to an increase of VCAM-1 promoter activity and the expression of VCAM-1 mRNA and protein in HPAEpiCs. Moreover, resveratrol can reduce lung inflammation via the inhibition of VCAM-1 expression, monocyte adhesion, and the activation of c-Src, PDGFR, JNK1/2, p38 MAPK, and AP-1 induced by S. aureus .

    Article Snippet: Anti-phospho-p38 MAPK, anti-phospho-JNK1/2, anti-phospho-p42/p44 MAPK, anti-phospho-c-Src, anti-phospho-PDGFR, anti-phospho-ATF2, and anti-phospho-c-Jun antibodies were obtained from Cell Signaling (Danver, MA, USA).

    Techniques: Expressing, Activation Assay, Activity Assay, Inhibition

    S. aureus induces VCAM-1 expression in HPAEpiCs via PDGFR. ( A ) Cells were transfected with scrambled, p42, p38, or JNK1 siRNA, and then incubated with S. aureus for 4 h, 6 h, or 24 h. The mRNA levels and promoter activity of VCAM-1 and the THP-1 cells adherence were measured. ( B ) Cells were pretreated without or with PP1 or AG1296 for 1 h, and then incubated with S. aureus for the indicated times. The expression of phospho-p38 MAPK, phospho-p42/p44 MAPK, and phospho-JNK1/2 were determined by Western blot. n = 3–4, # p

    Journal: International Journal of Molecular Sciences

    Article Title: Resveratrol Attenuates Staphylococcus Aureus-Induced Monocyte Adhesion through Downregulating PDGFR/AP-1 Activation in Human Lung Epithelial Cells

    doi: 10.3390/ijms19103058

    Figure Lengend Snippet: S. aureus induces VCAM-1 expression in HPAEpiCs via PDGFR. ( A ) Cells were transfected with scrambled, p42, p38, or JNK1 siRNA, and then incubated with S. aureus for 4 h, 6 h, or 24 h. The mRNA levels and promoter activity of VCAM-1 and the THP-1 cells adherence were measured. ( B ) Cells were pretreated without or with PP1 or AG1296 for 1 h, and then incubated with S. aureus for the indicated times. The expression of phospho-p38 MAPK, phospho-p42/p44 MAPK, and phospho-JNK1/2 were determined by Western blot. n = 3–4, # p

    Article Snippet: Anti-phospho-p38 MAPK, anti-phospho-JNK1/2, anti-phospho-p42/p44 MAPK, anti-phospho-c-Src, anti-phospho-PDGFR, anti-phospho-ATF2, and anti-phospho-c-Jun antibodies were obtained from Cell Signaling (Danver, MA, USA).

    Techniques: Expressing, Transfection, Incubation, Activity Assay, Western Blot

    Neuroprotection in hippocampal CA3 neurons, and the prevention of p-JNK, BACE1 and NOX2 induction by early continuous 17β-oestradiol (E2) replacement for 10 weeks in rats with LTED. ( A ) Representative staining of NeuN, TUNEL and amyloid-β1–42 of CA3 region from sham (Sh), and animals treated with 17β-oestradiol or placebo (Pla) immediately after ovariectomy for the entire 10-week period. The rats were subjected to sham or 10 min ischaemia followed by 7 days of reperfusion. ( B ) Cell-counting study shows the number of surviving neurons (% changes of sham) and TUNEL-positive cells per 250 µm length of medial CA3 region. Magnification: ×40. Scale bar = 50 µm. n = 6–7 in each group. # P

    Journal: Brain

    Article Title: Hypersensitivity of the hippocampal CA3 region to stress-induced neurodegeneration and amyloidogenesis in a rat model of surgical menopause

    doi: 10.1093/brain/awt046

    Figure Lengend Snippet: Neuroprotection in hippocampal CA3 neurons, and the prevention of p-JNK, BACE1 and NOX2 induction by early continuous 17β-oestradiol (E2) replacement for 10 weeks in rats with LTED. ( A ) Representative staining of NeuN, TUNEL and amyloid-β1–42 of CA3 region from sham (Sh), and animals treated with 17β-oestradiol or placebo (Pla) immediately after ovariectomy for the entire 10-week period. The rats were subjected to sham or 10 min ischaemia followed by 7 days of reperfusion. ( B ) Cell-counting study shows the number of surviving neurons (% changes of sham) and TUNEL-positive cells per 250 µm length of medial CA3 region. Magnification: ×40. Scale bar = 50 µm. n = 6–7 in each group. # P

    Article Snippet: The following primary antibodies were used in different combinations: anti-NeuN (1:500, MAB377, Millipore); anti-amyloid-β[1-42] (1:500, #700254) and anti-BACE1 (1:200, AHB0241) from Invitrogen Corporation; anti-phospho-JNK (1:50, sc-12882) and anti-phospho-cJun (1:50, sc-7981) from Santa Cruz Biotechnology, anti-phospho-amyloid precursor protein (1:400, #3823, Cell Signaling Technology), anti-PHF1 (1:2000; gift from Dr. Peter Davies).

    Techniques: Staining, TUNEL Assay, Proximity Ligation Assay, Cell Counting

    Activation and role of NOX2 NADPH oxidase and JNK/cJun signalling in CA3 neuronal cell death following LTED plus cerebral ischaemia. ( A ) Western blot analyses of NOX2 protein from CA3 samples at 3 h reperfusion in short/long term rats. ( B ) Representative microscopy images of NeuN (red) and NOX2 (green) in CA3 regions of placebo groups at 3 h ischaemic reperfusion. Magnification: ×40. Scale bar = 50 µm. ( C ) Changes of NADPH oxidase activity and superoxide production in CA3 region at 3 h reperfusion in the LTED animals. ( D and E ) Western blots of CA3 protein samples show activation of JNK and cJun induced by 3 h ischaemic reperfusion and the inhibition by the competitive NOX2 inhibitor gp91ds-tat peptide (91ds-tat) but not scrambled control peptide (Scr). ( F ) Administration of the JNK inhibitor SP600125 (SP) or 91ds-tat prevents cJun/AP-1 activity in CA3 samples at 3 h reperfusion in the LTED groups. ( G ) Quantitative data ( n = 5–7/group) showing the neuroprotective effects of 91ds-tat and SP in hippocampal CA3 region of rats with LTED at Day 7 of ischaemic reperfusion. A–F : n = 4–5 per group. * P

    Journal: Brain

    Article Title: Hypersensitivity of the hippocampal CA3 region to stress-induced neurodegeneration and amyloidogenesis in a rat model of surgical menopause

    doi: 10.1093/brain/awt046

    Figure Lengend Snippet: Activation and role of NOX2 NADPH oxidase and JNK/cJun signalling in CA3 neuronal cell death following LTED plus cerebral ischaemia. ( A ) Western blot analyses of NOX2 protein from CA3 samples at 3 h reperfusion in short/long term rats. ( B ) Representative microscopy images of NeuN (red) and NOX2 (green) in CA3 regions of placebo groups at 3 h ischaemic reperfusion. Magnification: ×40. Scale bar = 50 µm. ( C ) Changes of NADPH oxidase activity and superoxide production in CA3 region at 3 h reperfusion in the LTED animals. ( D and E ) Western blots of CA3 protein samples show activation of JNK and cJun induced by 3 h ischaemic reperfusion and the inhibition by the competitive NOX2 inhibitor gp91ds-tat peptide (91ds-tat) but not scrambled control peptide (Scr). ( F ) Administration of the JNK inhibitor SP600125 (SP) or 91ds-tat prevents cJun/AP-1 activity in CA3 samples at 3 h reperfusion in the LTED groups. ( G ) Quantitative data ( n = 5–7/group) showing the neuroprotective effects of 91ds-tat and SP in hippocampal CA3 region of rats with LTED at Day 7 of ischaemic reperfusion. A–F : n = 4–5 per group. * P

    Article Snippet: The following primary antibodies were used in different combinations: anti-NeuN (1:500, MAB377, Millipore); anti-amyloid-β[1-42] (1:500, #700254) and anti-BACE1 (1:200, AHB0241) from Invitrogen Corporation; anti-phospho-JNK (1:50, sc-12882) and anti-phospho-cJun (1:50, sc-7981) from Santa Cruz Biotechnology, anti-phospho-amyloid precursor protein (1:400, #3823, Cell Signaling Technology), anti-PHF1 (1:2000; gift from Dr. Peter Davies).

    Techniques: Activation Assay, Western Blot, Microscopy, Activity Assay, Inhibition

    Activation of JNK pathway induces amyloid precursor protein (T668) phosphorylation and an increase of C-99/C-83 amyloid precursor protein ratio in rats with LTED, and hypersensitivity of LTED CA3 neurons extends to the Alzheimer’s disease-relevant insult, amyloid-β1–42. ( A ) Representative confocal analysis shows increased immunoreactivity of p-APP (T668) and p-JNK in LTED CA3 neurons. ( B ) Western blot analyses of p-APP (T668) and total amyloid precursor protein in CA3 protein samples. ( C ) Western blot analysis specific for C-terminal fragment of amyloid precursor protein after α-secretase cleavage (C-83) and β-secretase cleavage (C-99). Ratios of C-99 fragments to C-83 fragments were expressed as fold changes versus the value in the short-term sham. JNK inhibition provoked a significant reduction of the C-99/C-83 ratio, indicating a shift towards the non-amyloidogenic pathway. ( D ) ELISA assay of amyloid-β1–42 and amyloid-β1–40 was performed by using hippocampal CA3 homogenates at 3 h ischaemic reperfusion ( a , b ). ( E ) Typical microscopy images showing the double staining of amyloid-β1–42 and TUNEL in CA3 region at 7 days reperfusion. Note that amyloid-β1–42 significantly merged with TUNEL-positive apoptotic-like CA3 neurons in rats with LTED. ( F ) Hypersensitivity of hippocampal CA3 neurons to amyloid-β1–42-induced neurotoxicity in rats with LTED. ( a ) Typical staining of NeuN in short/long-term 17β-oestradiol (E2) deprived rats at day 7 after infusion with amyloid-β1–42 or scrambled amyloid-β1–42 into the CA3 region of the hippocampus. ( b ) Quantitative analysis of data shows the numbers of surviving neurons in CA3 region. Scale bar = 50 µm. Magnification: ×40. A–E : n = 4–6 per group. * P

    Journal: Brain

    Article Title: Hypersensitivity of the hippocampal CA3 region to stress-induced neurodegeneration and amyloidogenesis in a rat model of surgical menopause

    doi: 10.1093/brain/awt046

    Figure Lengend Snippet: Activation of JNK pathway induces amyloid precursor protein (T668) phosphorylation and an increase of C-99/C-83 amyloid precursor protein ratio in rats with LTED, and hypersensitivity of LTED CA3 neurons extends to the Alzheimer’s disease-relevant insult, amyloid-β1–42. ( A ) Representative confocal analysis shows increased immunoreactivity of p-APP (T668) and p-JNK in LTED CA3 neurons. ( B ) Western blot analyses of p-APP (T668) and total amyloid precursor protein in CA3 protein samples. ( C ) Western blot analysis specific for C-terminal fragment of amyloid precursor protein after α-secretase cleavage (C-83) and β-secretase cleavage (C-99). Ratios of C-99 fragments to C-83 fragments were expressed as fold changes versus the value in the short-term sham. JNK inhibition provoked a significant reduction of the C-99/C-83 ratio, indicating a shift towards the non-amyloidogenic pathway. ( D ) ELISA assay of amyloid-β1–42 and amyloid-β1–40 was performed by using hippocampal CA3 homogenates at 3 h ischaemic reperfusion ( a , b ). ( E ) Typical microscopy images showing the double staining of amyloid-β1–42 and TUNEL in CA3 region at 7 days reperfusion. Note that amyloid-β1–42 significantly merged with TUNEL-positive apoptotic-like CA3 neurons in rats with LTED. ( F ) Hypersensitivity of hippocampal CA3 neurons to amyloid-β1–42-induced neurotoxicity in rats with LTED. ( a ) Typical staining of NeuN in short/long-term 17β-oestradiol (E2) deprived rats at day 7 after infusion with amyloid-β1–42 or scrambled amyloid-β1–42 into the CA3 region of the hippocampus. ( b ) Quantitative analysis of data shows the numbers of surviving neurons in CA3 region. Scale bar = 50 µm. Magnification: ×40. A–E : n = 4–6 per group. * P

    Article Snippet: The following primary antibodies were used in different combinations: anti-NeuN (1:500, MAB377, Millipore); anti-amyloid-β[1-42] (1:500, #700254) and anti-BACE1 (1:200, AHB0241) from Invitrogen Corporation; anti-phospho-JNK (1:50, sc-12882) and anti-phospho-cJun (1:50, sc-7981) from Santa Cruz Biotechnology, anti-phospho-amyloid precursor protein (1:400, #3823, Cell Signaling Technology), anti-PHF1 (1:2000; gift from Dr. Peter Davies).

    Techniques: Activation Assay, Western Blot, Inhibition, Enzyme-linked Immunosorbent Assay, Microscopy, Double Staining, TUNEL Assay, Staining

    BACE1 protein induction is mediated by activation of JNK/cJun signalling following ischaemic reperfusion in rats with LTED. ( A ) Western blot analyses of BACE1 and β-actin were performed with hippocampal CA3 protein in the indicated groups. BACE1 protein elevation induced by 3 h ischaemic reperfusion in rats with LTED was decreased by the JNK inhibitor SP600125 (SP). ( B ) Representative confocal analysis demonstrates increased immunofluorescence of BACE1 expression and active JNK/cJun in CA3 neurons in rats with LTED at 3 h reperfusion. Note that increased p-JNK/p-cJun staining co-localized well in the same cells with BACE1 protein in rats with LTED. ( C ) BACE1 activity in the indicated CA3 homogenates was assessed by measuring the liberated fluorescent fragment based on the BACE1-cleavable peptide substrate. ( D and E ) Representative sections of in situ hybridization for messenger RNA expression of BACE1 from sham ( a ), placebo ( b ), vehicle ( c ) and SP600125 ( d ) treated rats with LTED. Quantitative analysis of BACE1 messenger RNA expression in the CA3 pyramidal cell layer. Data are expressed as means ± SE from four to five rats per group, and shown as fold-change or percent-change versus the value in the short-term sham. * P

    Journal: Brain

    Article Title: Hypersensitivity of the hippocampal CA3 region to stress-induced neurodegeneration and amyloidogenesis in a rat model of surgical menopause

    doi: 10.1093/brain/awt046

    Figure Lengend Snippet: BACE1 protein induction is mediated by activation of JNK/cJun signalling following ischaemic reperfusion in rats with LTED. ( A ) Western blot analyses of BACE1 and β-actin were performed with hippocampal CA3 protein in the indicated groups. BACE1 protein elevation induced by 3 h ischaemic reperfusion in rats with LTED was decreased by the JNK inhibitor SP600125 (SP). ( B ) Representative confocal analysis demonstrates increased immunofluorescence of BACE1 expression and active JNK/cJun in CA3 neurons in rats with LTED at 3 h reperfusion. Note that increased p-JNK/p-cJun staining co-localized well in the same cells with BACE1 protein in rats with LTED. ( C ) BACE1 activity in the indicated CA3 homogenates was assessed by measuring the liberated fluorescent fragment based on the BACE1-cleavable peptide substrate. ( D and E ) Representative sections of in situ hybridization for messenger RNA expression of BACE1 from sham ( a ), placebo ( b ), vehicle ( c ) and SP600125 ( d ) treated rats with LTED. Quantitative analysis of BACE1 messenger RNA expression in the CA3 pyramidal cell layer. Data are expressed as means ± SE from four to five rats per group, and shown as fold-change or percent-change versus the value in the short-term sham. * P

    Article Snippet: The following primary antibodies were used in different combinations: anti-NeuN (1:500, MAB377, Millipore); anti-amyloid-β[1-42] (1:500, #700254) and anti-BACE1 (1:200, AHB0241) from Invitrogen Corporation; anti-phospho-JNK (1:50, sc-12882) and anti-phospho-cJun (1:50, sc-7981) from Santa Cruz Biotechnology, anti-phospho-amyloid precursor protein (1:400, #3823, Cell Signaling Technology), anti-PHF1 (1:2000; gift from Dr. Peter Davies).

    Techniques: Activation Assay, Western Blot, Immunofluorescence, Expressing, Staining, Activity Assay, In Situ Hybridization, RNA Expression

    Summary diagram of the proposed mechanisms underlying CA3 hypersensitivity, Alzheimer’s disease protein induction, neuronal death and behavioural deficits following cerebral ischaemia in rats with LTED. LTED plus stress (cerebral ischaemia) leads to hyperinduction of NOX2 NADPH oxidase activity and superoxide elevation, resulting in the activation of stress-responsive JNK/cJun signalling pathway in hippocampal CA3 neurons. JNK pathway activation induces the gene expression of BACE1 as well as amyloid precursor protein (Thr668) phosphorylation, which together promotes amyloid-β expression following ischaemic stress. JNK/cJun activation also initiates a positive-feedback regulation of NADPH oxidase and superoxide production by inducing NOX2 expression. Finally, Tau hyperphosphorylation resulting from JNK activation, together with amyloid-β generation and other possible factors induced by JNK/cJun signalling, leads to enhanced CA3 neuronal cell death and memory deterioration following cerebral ischaemia in rats with LTED. APP = amyloid precursor protein.

    Journal: Brain

    Article Title: Hypersensitivity of the hippocampal CA3 region to stress-induced neurodegeneration and amyloidogenesis in a rat model of surgical menopause

    doi: 10.1093/brain/awt046

    Figure Lengend Snippet: Summary diagram of the proposed mechanisms underlying CA3 hypersensitivity, Alzheimer’s disease protein induction, neuronal death and behavioural deficits following cerebral ischaemia in rats with LTED. LTED plus stress (cerebral ischaemia) leads to hyperinduction of NOX2 NADPH oxidase activity and superoxide elevation, resulting in the activation of stress-responsive JNK/cJun signalling pathway in hippocampal CA3 neurons. JNK pathway activation induces the gene expression of BACE1 as well as amyloid precursor protein (Thr668) phosphorylation, which together promotes amyloid-β expression following ischaemic stress. JNK/cJun activation also initiates a positive-feedback regulation of NADPH oxidase and superoxide production by inducing NOX2 expression. Finally, Tau hyperphosphorylation resulting from JNK activation, together with amyloid-β generation and other possible factors induced by JNK/cJun signalling, leads to enhanced CA3 neuronal cell death and memory deterioration following cerebral ischaemia in rats with LTED. APP = amyloid precursor protein.

    Article Snippet: The following primary antibodies were used in different combinations: anti-NeuN (1:500, MAB377, Millipore); anti-amyloid-β[1-42] (1:500, #700254) and anti-BACE1 (1:200, AHB0241) from Invitrogen Corporation; anti-phospho-JNK (1:50, sc-12882) and anti-phospho-cJun (1:50, sc-7981) from Santa Cruz Biotechnology, anti-phospho-amyloid precursor protein (1:400, #3823, Cell Signaling Technology), anti-PHF1 (1:2000; gift from Dr. Peter Davies).

    Techniques: Activity Assay, Activation Assay, Expressing

    Effects of CoPP treatment on the protein levels of Nrf2, HO-1, NQO1 and JNK in the sciatic nerve of diabetic mice. The protein levels of Nrf2 ( A ), HO-1 ( B ), NQO1 ( C ) and JNK ( D ) in the sciatic nerve from db/db mice treated with CoPP or vehicle and in db/+ mice treated with vehicle are represented. For each protein, * indicates significant differences as compared to db/+ mice treated with vehicle and # indicates significant differences vs. db/db mice treated with CoPP ( p

    Journal: International Journal of Molecular Sciences

    Article Title: The Inhibitory Effects of Cobalt Protoporphyrin IX and Cannabinoid 2 Receptor Agonists in Type 2 Diabetic Mice

    doi: 10.3390/ijms18112268

    Figure Lengend Snippet: Effects of CoPP treatment on the protein levels of Nrf2, HO-1, NQO1 and JNK in the sciatic nerve of diabetic mice. The protein levels of Nrf2 ( A ), HO-1 ( B ), NQO1 ( C ) and JNK ( D ) in the sciatic nerve from db/db mice treated with CoPP or vehicle and in db/+ mice treated with vehicle are represented. For each protein, * indicates significant differences as compared to db/+ mice treated with vehicle and # indicates significant differences vs. db/db mice treated with CoPP ( p

    Article Snippet: Proteins were electrophoretically transferred onto PVDF membrane for 120 min, blocked with PBS or TBST + 5% nonfat dry milk or TBST + 5% BSA and then incubated at 4 °C overnight with a rabbit anti-Nrf2 (1:160, Abcam, Cambridge, UK), anti-HO-1 (1:300, Abcam, Cambridge, UK), anti-NQO1 (1:350, Sigma, St. Louis, MO, USA), anti-phosphorylated JNK (1:250, Cell Signaling Technology (Danvers, MA, USA), anti-total JNK (1:250, Cell Signaling Technology (Danvers, MA, USA) and anti-CB2R (1:500, Abcam, Cambridge, UK) antibodies.

    Techniques: Mouse Assay

    Effects of GSI treatment on MAPK, PI3K/AKT and NF-κB pathway activation in LPS/IC-activated macrophages. (A-B) IFNγ-primed BMMs were pre-treated with vehicle control DMSO or GSI (25 μM) for 30 min and activated by LPS/ICs for 5, 15, 30 and 60 min. Phospho-p38, p38 phospho-p44-42, p44-42, phospho-SAPK/JNK, SAPK/JNK, phospho-Akt, Akt and the loading control β-actin were detected by Western blotting. Representative data from 1 of 3 independent experiments are shown. (C) IFNγ-primed BMMs were activated by LPS/ICs for 4 hrs in the presence of vehicle control DMSO or GSI (25 μM). NF-κB p50 was detected by immunofluorescence staining. The arrows indicate cells with decreased or no p50 nuclear translocation (green). Representative data from 1 of 2 independent experiments are shown.

    Journal: PLoS ONE

    Article Title: Notch signaling regulates the responses of lipopolysaccharide-stimulated macrophages in the presence of immune complexes

    doi: 10.1371/journal.pone.0198609

    Figure Lengend Snippet: Effects of GSI treatment on MAPK, PI3K/AKT and NF-κB pathway activation in LPS/IC-activated macrophages. (A-B) IFNγ-primed BMMs were pre-treated with vehicle control DMSO or GSI (25 μM) for 30 min and activated by LPS/ICs for 5, 15, 30 and 60 min. Phospho-p38, p38 phospho-p44-42, p44-42, phospho-SAPK/JNK, SAPK/JNK, phospho-Akt, Akt and the loading control β-actin were detected by Western blotting. Representative data from 1 of 3 independent experiments are shown. (C) IFNγ-primed BMMs were activated by LPS/ICs for 4 hrs in the presence of vehicle control DMSO or GSI (25 μM). NF-κB p50 was detected by immunofluorescence staining. The arrows indicate cells with decreased or no p50 nuclear translocation (green). Representative data from 1 of 2 independent experiments are shown.

    Article Snippet: The primary antibodies used in this study were as follows: rabbit anti-Notch1 (1:2000) (Santa Cruz Biotechnology, USA), rabbit anti-cleaved Notch1 (Val1744) (1:1000), rabbit anti-phospho-p38 (1:2000), rabbit anti p38 (1:2000), rabbit anti-phospho-p44-42 (1:4000), rabbit anti p44-42 (1:4000), rabbit anti-phospho-SAPK-JNK (1:2000), rabbit anti-SAPK-JNK (1:2000), rabbit anti-phospho-AKT (1:2000), rabbit anti-AKT (1:2000) and rabbit anti-RBPJSHU (1:1000) (all from Cell Signaling Technology, USA), mouse anti β-actin (1:1000) (Chemicon-Millipore, USA) and rabbit anti-GAPDH (1:4000) (Santa Cruz Biotechnology, USA).

    Techniques: Activation Assay, Western Blot, Immunofluorescence, Staining, Translocation Assay