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  • 99
    Cell Signaling Technology Inc phospho p38
    The <t>p38–ATF-7</t> Innate Immunity Pathway is Required for DR Longevity (A–C) DR lifespan extension is blocked by the p38 pathway loss-of-function mutations sek-1(km4) (A), nsy-1(ok593) (B), and pmk-1(km25) (C). (D) Lack of SEK-1 abolished lifespan extension by the feeding-impairment mutation eat-2 ). (E, F) The upstream immunity regulator TIR-1 is largely required in DR. Lifespan extension was reduced considerably by a pathogen-sensitive tir-1 mutant ( qd4 ), but only modestly by a tir-1 allele (ok1052) ). (G, H) Impairment of DR by the atf-7 loss-of-function ( lf ) allele atf-7(qd22 qd130lf) (G), and the gain-of-function constitutive repressor mutation atf-7(qd22gf) (H). ), had only a modest effect on DR. tph-1(n4622) is a presumed null deletion mutant. (J–L) DR lifespan extension was limited in extrachromosomal transgenic strains in which sek-1 ). Ad libitum (AL) food concentration is OD600 at 3 and DR is OD600 at 0.1. Unless otherwise specified, all AL/DR experiments were performed in liquid culture by dilution of growth-arrested bacterial food, and lifespans were measured at 20°C from hatching. The percent to which DR Increased lifespan (%, DR vs. .
    Phospho P38, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 7489 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc phospho p38 mapk
    MTD activate PMN Human PMN exposed to human MTD (muscle) were immunoblotted for phosphorylated and total (control) <t>p38</t> ( a ) or p44/42 <t>MAPK</t> ( b). MMP-8 was immunoblotted in supernatants ( c , d are from same gel). αFPR1 denotes anti-FPR1. e–f, MTD elicits PMN IL-8 synthesis: */** denote p
    Phospho P38 Mapk, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 3803 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc phosphorylated p38
    MTD activate PMN Human PMN exposed to human MTD (muscle) were immunoblotted for phosphorylated and total (control) <t>p38</t> ( a ) or p44/42 <t>MAPK</t> ( b). MMP-8 was immunoblotted in supernatants ( c , d are from same gel). αFPR1 denotes anti-FPR1. e–f, MTD elicits PMN IL-8 synthesis: */** denote p
    Phosphorylated P38, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1312 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti phospho p38 mapk
    MTD activate PMN Human PMN exposed to human MTD (muscle) were immunoblotted for phosphorylated and total (control) <t>p38</t> ( a ) or p44/42 <t>MAPK</t> ( b). MMP-8 was immunoblotted in supernatants ( c , d are from same gel). αFPR1 denotes anti-FPR1. e–f, MTD elicits PMN IL-8 synthesis: */** denote p
    Anti Phospho P38 Mapk, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1271 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc phosphorylated p38 mapk
    MTD activate PMN Human PMN exposed to human MTD (muscle) were immunoblotted for phosphorylated and total (control) <t>p38</t> ( a ) or p44/42 <t>MAPK</t> ( b). MMP-8 was immunoblotted in supernatants ( c , d are from same gel). αFPR1 denotes anti-FPR1. e–f, MTD elicits PMN IL-8 synthesis: */** denote p
    Phosphorylated P38 Mapk, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 629 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology phospho p38
    Blockage of NF-κB p65 and/or MAPK <t>p38</t> attenuates B7-H3-amplified proinflammatory cytokine and chemokine production in brain tissues of S . pneumoniae -infected mice. Mice were challenged with PBS as the control, live S . pneumoniae (SP), or live S . pneumoniae plus B7-H3 (SP+B7-H3) 1 hr after mice pretreated with the MAPK p38 inhibitor SB203580, the NF-κB p65 inhibitor PDTC, or their combination (SB203580+PDTC) as described in the Methods. Brain samples were collected at 18 hrs after challenges for detecting protein levels ( B ) of TNF-α ( A ), IL-1β ( B ), IL-6 ( C ), and MCP-1 ( D ) by ELISA. Data are expressed as mean ± SD of five to six mice per time point and represent two separate experiments. ** p
    Phospho P38, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 521 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    p38  (Abcam)
    99
    Abcam p38
    Blockage of NF-κB p65 and/or MAPK <t>p38</t> attenuates B7-H3-amplified proinflammatory cytokine and chemokine production in brain tissues of S . pneumoniae -infected mice. Mice were challenged with PBS as the control, live S . pneumoniae (SP), or live S . pneumoniae plus B7-H3 (SP+B7-H3) 1 hr after mice pretreated with the MAPK p38 inhibitor SB203580, the NF-κB p65 inhibitor PDTC, or their combination (SB203580+PDTC) as described in the Methods. Brain samples were collected at 18 hrs after challenges for detecting protein levels ( B ) of TNF-α ( A ), IL-1β ( B ), IL-6 ( C ), and MCP-1 ( D ) by ELISA. Data are expressed as mean ± SD of five to six mice per time point and represent two separate experiments. ** p
    P38, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 879 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc phosphor p38
    Blockage of NF-κB p65 and/or MAPK <t>p38</t> attenuates B7-H3-amplified proinflammatory cytokine and chemokine production in brain tissues of S . pneumoniae -infected mice. Mice were challenged with PBS as the control, live S . pneumoniae (SP), or live S . pneumoniae plus B7-H3 (SP+B7-H3) 1 hr after mice pretreated with the MAPK p38 inhibitor SB203580, the NF-κB p65 inhibitor PDTC, or their combination (SB203580+PDTC) as described in the Methods. Brain samples were collected at 18 hrs after challenges for detecting protein levels ( B ) of TNF-α ( A ), IL-1β ( B ), IL-6 ( C ), and MCP-1 ( D ) by ELISA. Data are expressed as mean ± SD of five to six mice per time point and represent two separate experiments. ** p
    Phosphor P38, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 437 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology anti phospho p38
    Effect of <t>p38</t> kinase stimulated by DMP1 on mineralized nodule formation. MC3T3-E1 cells were grown in mineralization media in the presence of DMP1 or SB203580 and DMP1 for 7, 14, and 21 days. Mineralized nodules were assayed with von Kossa staining. DMP1 stimulated the formation of mineralized nodules at 14 and 21 days, although SB203580 inhibitor suppressed nodule formation.
    Anti Phospho P38, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 320 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Cell Signaling Technology Inc phospho p38 map kinase
    Effect of <t>p38</t> kinase stimulated by DMP1 on mineralized nodule formation. MC3T3-E1 cells were grown in mineralization media in the presence of DMP1 or SB203580 and DMP1 for 7, 14, and 21 days. Mineralized nodules were assayed with von Kossa staining. DMP1 stimulated the formation of mineralized nodules at 14 and 21 days, although SB203580 inhibitor suppressed nodule formation.
    Phospho P38 Map Kinase, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 285 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti p38
    Apoptosis and autophagy rates in Ti, Hy (400 µg/ml)+Ti, TWEAK+Ti, TWEAK+Hy+Ti, Anisomycin+Ti and Anisomycin+Hy+Ti groups are determined using flow cytometry. (A) TWEAK overexpression and <t>p38</t> MAPK activation by anisomycin were able to elevate the apoptosis rate in Ti particle-injured cells, even under Hy pretreatment. (B) TWEAK overexpression and p38 MAPK activation by anisomycin may enhance the autophagy rate in Ti particle-injured cells, even under pretreatment with Hy condition. Black, control; green, Ti; pink, Hy+Ti; light blue, TWEAK+Ti; yellow, TWEAK+Hy+Ti; blue, Anisomycin+Ti; orange, Anisomycin+Hy+Ti. Data are presented as the mean ± standard deviation. n=3. *P
    Anti P38, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 398 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    The p38–ATF-7 Innate Immunity Pathway is Required for DR Longevity (A–C) DR lifespan extension is blocked by the p38 pathway loss-of-function mutations sek-1(km4) (A), nsy-1(ok593) (B), and pmk-1(km25) (C). (D) Lack of SEK-1 abolished lifespan extension by the feeding-impairment mutation eat-2 ). (E, F) The upstream immunity regulator TIR-1 is largely required in DR. Lifespan extension was reduced considerably by a pathogen-sensitive tir-1 mutant ( qd4 ), but only modestly by a tir-1 allele (ok1052) ). (G, H) Impairment of DR by the atf-7 loss-of-function ( lf ) allele atf-7(qd22 qd130lf) (G), and the gain-of-function constitutive repressor mutation atf-7(qd22gf) (H). ), had only a modest effect on DR. tph-1(n4622) is a presumed null deletion mutant. (J–L) DR lifespan extension was limited in extrachromosomal transgenic strains in which sek-1 ). Ad libitum (AL) food concentration is OD600 at 3 and DR is OD600 at 0.1. Unless otherwise specified, all AL/DR experiments were performed in liquid culture by dilution of growth-arrested bacterial food, and lifespans were measured at 20°C from hatching. The percent to which DR Increased lifespan (%, DR vs. .

    Journal: Cell metabolism

    Article Title: Dietary Restriction Extends Lifespan Through Metabolic Regulation of Innate Immunity

    doi: 10.1016/j.cmet.2019.02.013

    Figure Lengend Snippet: The p38–ATF-7 Innate Immunity Pathway is Required for DR Longevity (A–C) DR lifespan extension is blocked by the p38 pathway loss-of-function mutations sek-1(km4) (A), nsy-1(ok593) (B), and pmk-1(km25) (C). (D) Lack of SEK-1 abolished lifespan extension by the feeding-impairment mutation eat-2 ). (E, F) The upstream immunity regulator TIR-1 is largely required in DR. Lifespan extension was reduced considerably by a pathogen-sensitive tir-1 mutant ( qd4 ), but only modestly by a tir-1 allele (ok1052) ). (G, H) Impairment of DR by the atf-7 loss-of-function ( lf ) allele atf-7(qd22 qd130lf) (G), and the gain-of-function constitutive repressor mutation atf-7(qd22gf) (H). ), had only a modest effect on DR. tph-1(n4622) is a presumed null deletion mutant. (J–L) DR lifespan extension was limited in extrachromosomal transgenic strains in which sek-1 ). Ad libitum (AL) food concentration is OD600 at 3 and DR is OD600 at 0.1. Unless otherwise specified, all AL/DR experiments were performed in liquid culture by dilution of growth-arrested bacterial food, and lifespans were measured at 20°C from hatching. The percent to which DR Increased lifespan (%, DR vs. .

    Article Snippet: Protein samples were boiled 95°C for 10 min. Western blot analysis was performe d under standard conditions with antibodies against phospho-p38 (Cell Signaling) or total PMK1 antibodies ( ).

    Techniques: Mutagenesis, Transgenic Assay, Concentration Assay

    Regulation of p38–ATF-7 immunity and DR longevity by nutrients (A, B) Dose-response effect of bacterial food on T24B8.5 immunity reporter expression. Fluorescence microscopy images (A) and quantification (B) indicate GFP expression from day-six-adult WT worms that carry the agIs219 transgene, and have been fed for three-days under different concentrations of antibiotic-arrested OP50 bacteria in liquid medium. (C) UV treatment of cold- and antibiotic-treated OP50 did not affect T24B8.5 reporter expression. (D) Expression of T24B8.5 in C. elegans nutrient rich medium (CeNRM), a liquid axenic medium. Worms were grown in CeNRM until the second day of adulthood (day 7 from the L1 stage), then transferred into fresh CeNRM (AL, 1X) or diluted medium (DR, 0.05X) for four days. (E) Effect of nutrients on T24B8.5 expression. Day-three-adults were transferred to DD (dietary deprivation) conditions that included the indicated CeNRM components. Fluorescence microscopy images show day-seven adults. Only milk powder was sufficient to drive immune gene expression. The presence of FUdR in the DD medium resulted in embryonic growth arrest and reporter expression in embryos (apparent as speckles). (F, G) Survival of wild type N2 and atf-7(gf) animals that had been fed with CeNRM (F) or milk powder (G). Lifespan extension from A6d-initiated DD (F) was suppressed by continued CeNRM feeding, and reduced to a lesser extent by feeding with 0.2X CeNRM. Similar effects were seen upon feeding with milk powder (G). (H) Effects of mTORC1 inhibition on p38–ATF-7-regulated immunity gene expression ( C17H12.8 , K08D8.5 , T24B8.5 ). raga-1 and ragc-1 ), and the cytoprotective gene gst-4 ). Day 3 adults were examined by qRT-PCR after initiation of RNAi at the L4 stage. Scale bar, 100 μm. Mean ± SEM, two-tailed t-test. * P

    Journal: Cell metabolism

    Article Title: Dietary Restriction Extends Lifespan Through Metabolic Regulation of Innate Immunity

    doi: 10.1016/j.cmet.2019.02.013

    Figure Lengend Snippet: Regulation of p38–ATF-7 immunity and DR longevity by nutrients (A, B) Dose-response effect of bacterial food on T24B8.5 immunity reporter expression. Fluorescence microscopy images (A) and quantification (B) indicate GFP expression from day-six-adult WT worms that carry the agIs219 transgene, and have been fed for three-days under different concentrations of antibiotic-arrested OP50 bacteria in liquid medium. (C) UV treatment of cold- and antibiotic-treated OP50 did not affect T24B8.5 reporter expression. (D) Expression of T24B8.5 in C. elegans nutrient rich medium (CeNRM), a liquid axenic medium. Worms were grown in CeNRM until the second day of adulthood (day 7 from the L1 stage), then transferred into fresh CeNRM (AL, 1X) or diluted medium (DR, 0.05X) for four days. (E) Effect of nutrients on T24B8.5 expression. Day-three-adults were transferred to DD (dietary deprivation) conditions that included the indicated CeNRM components. Fluorescence microscopy images show day-seven adults. Only milk powder was sufficient to drive immune gene expression. The presence of FUdR in the DD medium resulted in embryonic growth arrest and reporter expression in embryos (apparent as speckles). (F, G) Survival of wild type N2 and atf-7(gf) animals that had been fed with CeNRM (F) or milk powder (G). Lifespan extension from A6d-initiated DD (F) was suppressed by continued CeNRM feeding, and reduced to a lesser extent by feeding with 0.2X CeNRM. Similar effects were seen upon feeding with milk powder (G). (H) Effects of mTORC1 inhibition on p38–ATF-7-regulated immunity gene expression ( C17H12.8 , K08D8.5 , T24B8.5 ). raga-1 and ragc-1 ), and the cytoprotective gene gst-4 ). Day 3 adults were examined by qRT-PCR after initiation of RNAi at the L4 stage. Scale bar, 100 μm. Mean ± SEM, two-tailed t-test. * P

    Article Snippet: Protein samples were boiled 95°C for 10 min. Western blot analysis was performe d under standard conditions with antibodies against phospho-p38 (Cell Signaling) or total PMK1 antibodies ( ).

    Techniques: Expressing, Fluorescence, Microscopy, Inhibition, Quantitative RT-PCR, Two Tailed Test

    rIIS and DAF-16 inhibit food consumption (A) The long-lived mutant daf-2(e1370) consumes reduced amounts of food. The relative consumption of food (antibiotic-arrested OP50 in liquid medium) was determined by the change in OD600 absorbance over time. (B, C) Effects of rIIS and DAF-16 on food intake. DAF-16 is required for rIIS to reduce food intake, and daf-16 mutants consume more food than WT animals. (D, E) Effects of rIIS and DAF-16 on food intake (D) and immunity gene expression (E) when animals were fed with the three E. coli strains most commonly used in the laboratory as C. elegans food sources. Here HT115 is carrying the L4440 plasmid. (F, G) Effect of daf-2 RNAi on expression of the p38–ATF-7 target gene T24B8.5 in daf-16 and atf-7(lf) mutants. Fluorescence microscopy images (F) and quantification (G) indicate GFP levels of day-one-adults in solid agar plates. (H) Three immunity genes ( C17H12.8 , K08D8.5 , T24B8.5 ) are downregulated by DD or the decreased feeding associated with rIIS, with this partially reversed by milk feeding. Expression was examined by qRT-PCR. (I, J) Effects of IIS, DD, and autoclaved milk powder on expression of the p38–ATF-7-regulated gene T24B8.5. Day-three-adults were transferred to DD medium, and fluorescence microscopy images (I) and quantification (J) indicate GFP levels after four days of DD treatment (day-seven adult). Scale bar, 100 μm. Mean ± SEM, two-tailed t-test. * P

    Journal: Cell metabolism

    Article Title: Dietary Restriction Extends Lifespan Through Metabolic Regulation of Innate Immunity

    doi: 10.1016/j.cmet.2019.02.013

    Figure Lengend Snippet: rIIS and DAF-16 inhibit food consumption (A) The long-lived mutant daf-2(e1370) consumes reduced amounts of food. The relative consumption of food (antibiotic-arrested OP50 in liquid medium) was determined by the change in OD600 absorbance over time. (B, C) Effects of rIIS and DAF-16 on food intake. DAF-16 is required for rIIS to reduce food intake, and daf-16 mutants consume more food than WT animals. (D, E) Effects of rIIS and DAF-16 on food intake (D) and immunity gene expression (E) when animals were fed with the three E. coli strains most commonly used in the laboratory as C. elegans food sources. Here HT115 is carrying the L4440 plasmid. (F, G) Effect of daf-2 RNAi on expression of the p38–ATF-7 target gene T24B8.5 in daf-16 and atf-7(lf) mutants. Fluorescence microscopy images (F) and quantification (G) indicate GFP levels of day-one-adults in solid agar plates. (H) Three immunity genes ( C17H12.8 , K08D8.5 , T24B8.5 ) are downregulated by DD or the decreased feeding associated with rIIS, with this partially reversed by milk feeding. Expression was examined by qRT-PCR. (I, J) Effects of IIS, DD, and autoclaved milk powder on expression of the p38–ATF-7-regulated gene T24B8.5. Day-three-adults were transferred to DD medium, and fluorescence microscopy images (I) and quantification (J) indicate GFP levels after four days of DD treatment (day-seven adult). Scale bar, 100 μm. Mean ± SEM, two-tailed t-test. * P

    Article Snippet: Protein samples were boiled 95°C for 10 min. Western blot analysis was performe d under standard conditions with antibodies against phospho-p38 (Cell Signaling) or total PMK1 antibodies ( ).

    Techniques: Mutagenesis, Expressing, Plasmid Preparation, Fluorescence, Microscopy, Quantitative RT-PCR, Two Tailed Test

    Similar modulation of p38–ATF-7-mediated immunity by DR and rIIS (A – C) Comparison of DR and daf-2 RNAi effects on the survival of wild type (A), sek-1(km4) (B) and atf-7(qd22gf) (C) animals. (D, E) Effects of DR and sek-1 mutation on genes that are upregulated (D) or downregulated (E) in daf-2 ( − . These daf-2 ( − ). (F) Effects of DR and daf-2 RNAi on expression of p38–ATF-7-regulated immunity genes ( C17H12.8 , K08D8.5 and T24B8.5 ) in wild type and sek-1(km4) ), examined by qRT-PCR. DR was performed for three days. (G, H) Effects of DR and daf-2 RNAi on expression of the immune response gene T24B8.5, ). Fluorescence microscopy images (G) and quantification (H) indicate GFP levels at the indicated number of days after DR was initiated, or in parallel AL-fed controls. (I, J) DR and daf-2 RNAi suppress PMK-1 (p38) activity. Immunoblot analyses of total and activated (phosphorylated) PMK-1 from three-day RNAi-treated day-one-adult (I) and three-day DR treated worms (J) are shown. Scale bar, 100 μm. Mean ± SEM, two-tailed t-test. * P

    Journal: Cell metabolism

    Article Title: Dietary Restriction Extends Lifespan Through Metabolic Regulation of Innate Immunity

    doi: 10.1016/j.cmet.2019.02.013

    Figure Lengend Snippet: Similar modulation of p38–ATF-7-mediated immunity by DR and rIIS (A – C) Comparison of DR and daf-2 RNAi effects on the survival of wild type (A), sek-1(km4) (B) and atf-7(qd22gf) (C) animals. (D, E) Effects of DR and sek-1 mutation on genes that are upregulated (D) or downregulated (E) in daf-2 ( − . These daf-2 ( − ). (F) Effects of DR and daf-2 RNAi on expression of p38–ATF-7-regulated immunity genes ( C17H12.8 , K08D8.5 and T24B8.5 ) in wild type and sek-1(km4) ), examined by qRT-PCR. DR was performed for three days. (G, H) Effects of DR and daf-2 RNAi on expression of the immune response gene T24B8.5, ). Fluorescence microscopy images (G) and quantification (H) indicate GFP levels at the indicated number of days after DR was initiated, or in parallel AL-fed controls. (I, J) DR and daf-2 RNAi suppress PMK-1 (p38) activity. Immunoblot analyses of total and activated (phosphorylated) PMK-1 from three-day RNAi-treated day-one-adult (I) and three-day DR treated worms (J) are shown. Scale bar, 100 μm. Mean ± SEM, two-tailed t-test. * P

    Article Snippet: Protein samples were boiled 95°C for 10 min. Western blot analysis was performe d under standard conditions with antibodies against phospho-p38 (Cell Signaling) or total PMK1 antibodies ( ).

    Techniques: Mutagenesis, Expressing, Quantitative RT-PCR, Fluorescence, Microscopy, Activity Assay, Two Tailed Test

    DR Increases Cytoprotection but Inhibits p38-regulated Immunity Genes (A) Plan for RNA-seq experiments. Analysis of sek-1 overcame possible redundancy among the three PMK kinases. (B, C) Heatmap of DR-upregulated (B) and -downregulated (C) genes. DR-upregulated genes are defined as those genes having a higher expression under DR than AL conditions in WT animals. Throughout the manuscript, SEK-1 up- or down-regulated refers to genes that are expressed differentially between WT and sek-1(−) animals under both DR and AL conditions. SEK-1-upregulated genes that are upregulated (46 genes) or downregulated (162 genes) by DR are indicated with green boxes. A threshold of P

    Journal: Cell metabolism

    Article Title: Dietary Restriction Extends Lifespan Through Metabolic Regulation of Innate Immunity

    doi: 10.1016/j.cmet.2019.02.013

    Figure Lengend Snippet: DR Increases Cytoprotection but Inhibits p38-regulated Immunity Genes (A) Plan for RNA-seq experiments. Analysis of sek-1 overcame possible redundancy among the three PMK kinases. (B, C) Heatmap of DR-upregulated (B) and -downregulated (C) genes. DR-upregulated genes are defined as those genes having a higher expression under DR than AL conditions in WT animals. Throughout the manuscript, SEK-1 up- or down-regulated refers to genes that are expressed differentially between WT and sek-1(−) animals under both DR and AL conditions. SEK-1-upregulated genes that are upregulated (46 genes) or downregulated (162 genes) by DR are indicated with green boxes. A threshold of P

    Article Snippet: Protein samples were boiled 95°C for 10 min. Western blot analysis was performe d under standard conditions with antibodies against phospho-p38 (Cell Signaling) or total PMK1 antibodies ( ).

    Techniques: RNA Sequencing Assay, Expressing

    Modulation of p38–ATF-7 downstream gene expression determines lifespan (A, B) Regulation of T24B8.5 expression by ATF-7 and VHP-1. Fluorescence microscopy images (A) and quantification (B) indicate GFP expression from day-one-adult WT, atf-7(qd22 qd130lf) , and atf-7(qd22gf) worms that carry the agIs219 immunity reporter transgene, and have been subjected to two-day RNAi treatment. Scale bar, 100 μm. (C) The survival impairment induced by hyperactive p38 signaling ( vhp-1 RNAi) is ameliorated by atf-7 mutations. (D) DR and rIIS partially compensate for the lifespan reduction induced by vhp-1 RNAi. (E) Comparison of the effects of DR and daf-2 RNAi on T24B8.5 mRNA expression in WT and atf-7::GFP integrated transgenic animals after three days of liquid DR treatment, assayed by qRT-PCR. (F) Pathogenesis assay of day-one-adult worms exposed to P. aeruginosa PA14. Survival is enhanced or reduced by ATF-7 overexpression and loss-of-function, respectively. (G) DR and rIIS partially compensate for the lifespan reduction induced by transgenic ATF-7 overexpression. (H) Regulation of the p38 – ATF-7 immunometabolic pathway. Effects of DR and DAF-16-mediated reduced food intake on this pathway are shown in green. Scale bar, 100 μm. Mean ± SEM, two-tailed t-test. * P

    Journal: Cell metabolism

    Article Title: Dietary Restriction Extends Lifespan Through Metabolic Regulation of Innate Immunity

    doi: 10.1016/j.cmet.2019.02.013

    Figure Lengend Snippet: Modulation of p38–ATF-7 downstream gene expression determines lifespan (A, B) Regulation of T24B8.5 expression by ATF-7 and VHP-1. Fluorescence microscopy images (A) and quantification (B) indicate GFP expression from day-one-adult WT, atf-7(qd22 qd130lf) , and atf-7(qd22gf) worms that carry the agIs219 immunity reporter transgene, and have been subjected to two-day RNAi treatment. Scale bar, 100 μm. (C) The survival impairment induced by hyperactive p38 signaling ( vhp-1 RNAi) is ameliorated by atf-7 mutations. (D) DR and rIIS partially compensate for the lifespan reduction induced by vhp-1 RNAi. (E) Comparison of the effects of DR and daf-2 RNAi on T24B8.5 mRNA expression in WT and atf-7::GFP integrated transgenic animals after three days of liquid DR treatment, assayed by qRT-PCR. (F) Pathogenesis assay of day-one-adult worms exposed to P. aeruginosa PA14. Survival is enhanced or reduced by ATF-7 overexpression and loss-of-function, respectively. (G) DR and rIIS partially compensate for the lifespan reduction induced by transgenic ATF-7 overexpression. (H) Regulation of the p38 – ATF-7 immunometabolic pathway. Effects of DR and DAF-16-mediated reduced food intake on this pathway are shown in green. Scale bar, 100 μm. Mean ± SEM, two-tailed t-test. * P

    Article Snippet: Protein samples were boiled 95°C for 10 min. Western blot analysis was performe d under standard conditions with antibodies against phospho-p38 (Cell Signaling) or total PMK1 antibodies ( ).

    Techniques: Expressing, Fluorescence, Microscopy, Transgenic Assay, Quantitative RT-PCR, Over Expression, Two Tailed Test

    Effect of ATV on HSP22, eNOS and p38 MAPK in HUVECs. The expression of HSP22 in HUVECs with an HFD and/or ATV treatment was measured by (A) reverse transcription-quantitative polymerase chain reaction and (B) western blot assay, respectively. (C and D) The expression levels of p-eNOS and p-p38 MAPK were examined by western blot assay in HUVECs with an HFD and/or ATV treatment. ** P

    Journal: International Journal of Molecular Medicine

    Article Title: Atorvastatin downregulates HSP22 expression in an atherosclerotic model in vitro and in vivo

    doi: 10.3892/ijmm.2018.4015

    Figure Lengend Snippet: Effect of ATV on HSP22, eNOS and p38 MAPK in HUVECs. The expression of HSP22 in HUVECs with an HFD and/or ATV treatment was measured by (A) reverse transcription-quantitative polymerase chain reaction and (B) western blot assay, respectively. (C and D) The expression levels of p-eNOS and p-p38 MAPK were examined by western blot assay in HUVECs with an HFD and/or ATV treatment. ** P

    Article Snippet: Following blocking with 2% bovine serum albumin (Sigma Aldrich; Merck KGaA) in PBS for 1 h at room temperature, the tissue sections were incubated with anti-HSP22 (1:100 dilution; cat. no. ab151552; Abcam, Cambridge, MA, USA) and anti-phosphorylated p38 (anti-p-p38) (1:1,600; cat. no. 9212; Cell Signaling Technology, Inc., Danvers, MA, USA) primary antibodies at 4°C overnight, and then incubated with biotinylated secondary antibodies, horseradish peroxidase goat anti-rabbit IgG (1:100 dilution; cat. no. 111-035-008) and goat anti-mouse IgG (H+L) (1:100 dilution; cat. no. 111-035-003) (noth Jackson ImmunoResearch, West Grove, PA, USA) for 1 h at room temperature.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot

    HSP22-knockdown inhibits ox-LDL-induced p-eNOS decrease and p-p38 increase in HUVECs. (A) The transfection efficiency of HSP22 shRNAs in HUVECs was measured by western blot assay. (B) The expression of HSP22 in HUVECs with an HFD and/or ATV treatment was measured by western blot assay. (C and D) The expression of p-eNOS and p-p38 in HUVECs with an HFD and/or ATV treatment in the absence or presence of HSP22 shRNA was measured by western blot assay. * P

    Journal: International Journal of Molecular Medicine

    Article Title: Atorvastatin downregulates HSP22 expression in an atherosclerotic model in vitro and in vivo

    doi: 10.3892/ijmm.2018.4015

    Figure Lengend Snippet: HSP22-knockdown inhibits ox-LDL-induced p-eNOS decrease and p-p38 increase in HUVECs. (A) The transfection efficiency of HSP22 shRNAs in HUVECs was measured by western blot assay. (B) The expression of HSP22 in HUVECs with an HFD and/or ATV treatment was measured by western blot assay. (C and D) The expression of p-eNOS and p-p38 in HUVECs with an HFD and/or ATV treatment in the absence or presence of HSP22 shRNA was measured by western blot assay. * P

    Article Snippet: Following blocking with 2% bovine serum albumin (Sigma Aldrich; Merck KGaA) in PBS for 1 h at room temperature, the tissue sections were incubated with anti-HSP22 (1:100 dilution; cat. no. ab151552; Abcam, Cambridge, MA, USA) and anti-phosphorylated p38 (anti-p-p38) (1:1,600; cat. no. 9212; Cell Signaling Technology, Inc., Danvers, MA, USA) primary antibodies at 4°C overnight, and then incubated with biotinylated secondary antibodies, horseradish peroxidase goat anti-rabbit IgG (1:100 dilution; cat. no. 111-035-008) and goat anti-mouse IgG (H+L) (1:100 dilution; cat. no. 111-035-003) (noth Jackson ImmunoResearch, West Grove, PA, USA) for 1 h at room temperature.

    Techniques: Transfection, Western Blot, Expressing, shRNA

    Effect of HFD on HSP22, eNOS and p38 MAPK in atherosclerotic lesions. The expression of HSP22 in ApoE −/− mice with an HFD and/or ATV treatment was measured by (A) immunohistochemistry, (B) enzyme-linked immunosorbent assay and (C and D) western blot assay, respectively. (C and E) The expression level of p-eNOS was examined by western blot assay in ApoE −/− mice with an HFD and/or ATV treatment. The expression level of p-p38 MAPK was examined by (C and E) western blot assay and (F) immunohistochemistry in ApoE −/− mice with an HFD and/or ATV treatment. Data are presented as the mean ± standard deviation (n=6). ** P

    Journal: International Journal of Molecular Medicine

    Article Title: Atorvastatin downregulates HSP22 expression in an atherosclerotic model in vitro and in vivo

    doi: 10.3892/ijmm.2018.4015

    Figure Lengend Snippet: Effect of HFD on HSP22, eNOS and p38 MAPK in atherosclerotic lesions. The expression of HSP22 in ApoE −/− mice with an HFD and/or ATV treatment was measured by (A) immunohistochemistry, (B) enzyme-linked immunosorbent assay and (C and D) western blot assay, respectively. (C and E) The expression level of p-eNOS was examined by western blot assay in ApoE −/− mice with an HFD and/or ATV treatment. The expression level of p-p38 MAPK was examined by (C and E) western blot assay and (F) immunohistochemistry in ApoE −/− mice with an HFD and/or ATV treatment. Data are presented as the mean ± standard deviation (n=6). ** P

    Article Snippet: Following blocking with 2% bovine serum albumin (Sigma Aldrich; Merck KGaA) in PBS for 1 h at room temperature, the tissue sections were incubated with anti-HSP22 (1:100 dilution; cat. no. ab151552; Abcam, Cambridge, MA, USA) and anti-phosphorylated p38 (anti-p-p38) (1:1,600; cat. no. 9212; Cell Signaling Technology, Inc., Danvers, MA, USA) primary antibodies at 4°C overnight, and then incubated with biotinylated secondary antibodies, horseradish peroxidase goat anti-rabbit IgG (1:100 dilution; cat. no. 111-035-008) and goat anti-mouse IgG (H+L) (1:100 dilution; cat. no. 111-035-003) (noth Jackson ImmunoResearch, West Grove, PA, USA) for 1 h at room temperature.

    Techniques: Expressing, Mouse Assay, Immunohistochemistry, Enzyme-linked Immunosorbent Assay, Western Blot, Standard Deviation

    p38 MAPK inactivation attenuates scratch-induced astrogliosis in primary astrocyte cultures

    Journal: Brain research

    Article Title: INVOLVEMENT OF P38 MAPK IN REACTIVE ASTROGLIOSIS INDUCED BY ISCHEMIC STROKE

    doi: 10.1016/j.brainres.2014.01.013

    Figure Lengend Snippet: p38 MAPK inactivation attenuates scratch-induced astrogliosis in primary astrocyte cultures

    Article Snippet: Sections were stained with primary antibodies for GFAP (Santa Cruz, 1:100), p38 (Cell signaling, 1:100), Phoshporylated p38 (Cell signaling, 1:100) followed by secondary poly horse radish peroxidase (HRP) and 3,3'-Diaminobenzidine (DAB) staining.

    Techniques:

    Astrocyte p38 MAPK knockout has no effect on motor function recovery and lesion volume at acute stage after permanent MCAO in mice

    Journal: Brain research

    Article Title: INVOLVEMENT OF P38 MAPK IN REACTIVE ASTROGLIOSIS INDUCED BY ISCHEMIC STROKE

    doi: 10.1016/j.brainres.2014.01.013

    Figure Lengend Snippet: Astrocyte p38 MAPK knockout has no effect on motor function recovery and lesion volume at acute stage after permanent MCAO in mice

    Article Snippet: Sections were stained with primary antibodies for GFAP (Santa Cruz, 1:100), p38 (Cell signaling, 1:100), Phoshporylated p38 (Cell signaling, 1:100) followed by secondary poly horse radish peroxidase (HRP) and 3,3'-Diaminobenzidine (DAB) staining.

    Techniques: Knock-Out, Mouse Assay

    p38 MAPK inactivation inhibits primary astrocyte migration without affecting cell proliferation

    Journal: Brain research

    Article Title: INVOLVEMENT OF P38 MAPK IN REACTIVE ASTROGLIOSIS INDUCED BY ISCHEMIC STROKE

    doi: 10.1016/j.brainres.2014.01.013

    Figure Lengend Snippet: p38 MAPK inactivation inhibits primary astrocyte migration without affecting cell proliferation

    Article Snippet: Sections were stained with primary antibodies for GFAP (Santa Cruz, 1:100), p38 (Cell signaling, 1:100), Phoshporylated p38 (Cell signaling, 1:100) followed by secondary poly horse radish peroxidase (HRP) and 3,3'-Diaminobenzidine (DAB) staining.

    Techniques: Migration

    p38 MAPK inhibition attenuates OGD induced astrogliosis in primary astrocyte culture

    Journal: Brain research

    Article Title: INVOLVEMENT OF P38 MAPK IN REACTIVE ASTROGLIOSIS INDUCED BY ISCHEMIC STROKE

    doi: 10.1016/j.brainres.2014.01.013

    Figure Lengend Snippet: p38 MAPK inhibition attenuates OGD induced astrogliosis in primary astrocyte culture

    Article Snippet: Sections were stained with primary antibodies for GFAP (Santa Cruz, 1:100), p38 (Cell signaling, 1:100), Phoshporylated p38 (Cell signaling, 1:100) followed by secondary poly horse radish peroxidase (HRP) and 3,3'-Diaminobenzidine (DAB) staining.

    Techniques: Inhibition

    p38 MAPK knockout attenuates astrogliosis in primary astrocyte cultures

    Journal: Brain research

    Article Title: INVOLVEMENT OF P38 MAPK IN REACTIVE ASTROGLIOSIS INDUCED BY ISCHEMIC STROKE

    doi: 10.1016/j.brainres.2014.01.013

    Figure Lengend Snippet: p38 MAPK knockout attenuates astrogliosis in primary astrocyte cultures

    Article Snippet: Sections were stained with primary antibodies for GFAP (Santa Cruz, 1:100), p38 (Cell signaling, 1:100), Phoshporylated p38 (Cell signaling, 1:100) followed by secondary poly horse radish peroxidase (HRP) and 3,3'-Diaminobenzidine (DAB) staining.

    Techniques: Knock-Out

    p38 MAPK inactivation alters cytokine expression in primary astrocyte cultures

    Journal: Brain research

    Article Title: INVOLVEMENT OF P38 MAPK IN REACTIVE ASTROGLIOSIS INDUCED BY ISCHEMIC STROKE

    doi: 10.1016/j.brainres.2014.01.013

    Figure Lengend Snippet: p38 MAPK inactivation alters cytokine expression in primary astrocyte cultures

    Article Snippet: Sections were stained with primary antibodies for GFAP (Santa Cruz, 1:100), p38 (Cell signaling, 1:100), Phoshporylated p38 (Cell signaling, 1:100) followed by secondary poly horse radish peroxidase (HRP) and 3,3'-Diaminobenzidine (DAB) staining.

    Techniques: Expressing

    p38 MAPK inactivation attenuates wound healing in primary astrocyte cultures

    Journal: Brain research

    Article Title: INVOLVEMENT OF P38 MAPK IN REACTIVE ASTROGLIOSIS INDUCED BY ISCHEMIC STROKE

    doi: 10.1016/j.brainres.2014.01.013

    Figure Lengend Snippet: p38 MAPK inactivation attenuates wound healing in primary astrocyte cultures

    Article Snippet: Sections were stained with primary antibodies for GFAP (Santa Cruz, 1:100), p38 (Cell signaling, 1:100), Phoshporylated p38 (Cell signaling, 1:100) followed by secondary poly horse radish peroxidase (HRP) and 3,3'-Diaminobenzidine (DAB) staining.

    Techniques:

    Increase of GFAP and p38 expression in the peri-infarct region after ischemic stroke

    Journal: Brain research

    Article Title: INVOLVEMENT OF P38 MAPK IN REACTIVE ASTROGLIOSIS INDUCED BY ISCHEMIC STROKE

    doi: 10.1016/j.brainres.2014.01.013

    Figure Lengend Snippet: Increase of GFAP and p38 expression in the peri-infarct region after ischemic stroke

    Article Snippet: Sections were stained with primary antibodies for GFAP (Santa Cruz, 1:100), p38 (Cell signaling, 1:100), Phoshporylated p38 (Cell signaling, 1:100) followed by secondary poly horse radish peroxidase (HRP) and 3,3'-Diaminobenzidine (DAB) staining.

    Techniques: Expressing

    MTD activate PMN Human PMN exposed to human MTD (muscle) were immunoblotted for phosphorylated and total (control) p38 ( a ) or p44/42 MAPK ( b). MMP-8 was immunoblotted in supernatants ( c , d are from same gel). αFPR1 denotes anti-FPR1. e–f, MTD elicits PMN IL-8 synthesis: */** denote p

    Journal: Nature

    Article Title: Circulating Mitochondrial DAMPs Cause Inflammatory Responses to Injury

    doi: 10.1038/nature08780

    Figure Lengend Snippet: MTD activate PMN Human PMN exposed to human MTD (muscle) were immunoblotted for phosphorylated and total (control) p38 ( a ) or p44/42 MAPK ( b). MMP-8 was immunoblotted in supernatants ( c , d are from same gel). αFPR1 denotes anti-FPR1. e–f, MTD elicits PMN IL-8 synthesis: */** denote p

    Article Snippet: Antibodies to phospho-p38 MAPK (Thr180/Tyr182), p38 MAPK, phospho-p44/42 MAPK (Thr202/Tyr204) and p44/42 MAPK were from Cell Signaling (Danvers, MA).

    Techniques:

    mtDNA activates PMN via CpG/TLR9 interactions a, Incubation of PMN (10 6 ) with 1μg/ml mtDNA activates p38 MAPK (n=3, *p

    Journal: Nature

    Article Title: Circulating Mitochondrial DAMPs Cause Inflammatory Responses to Injury

    doi: 10.1038/nature08780

    Figure Lengend Snippet: mtDNA activates PMN via CpG/TLR9 interactions a, Incubation of PMN (10 6 ) with 1μg/ml mtDNA activates p38 MAPK (n=3, *p

    Article Snippet: Antibodies to phospho-p38 MAPK (Thr180/Tyr182), p38 MAPK, phospho-p44/42 MAPK (Thr202/Tyr204) and p44/42 MAPK were from Cell Signaling (Danvers, MA).

    Techniques: Incubation

    Hog1 is required for nitrosative-stress resistance. (A) To assay stress resistance, the following C. albicans strains were spotted in 10-fold serial dilutions on plates containing no stress (control), 25 mM succinic acid (SA), 5 mM NaNO 2 , 25 mM succinic acid plus 5 mM NaNO 2 , 0.01 mM NaOH, or 2.5 mM DPTA-NONOate in 0.01 mM NaOH: RM1000+CIp20 ( HOG1/HOG1 ), Ca2226 ( HOG1/hog1 ), JC50 ( hog1/hog1 ), and JC76 ( hog1 AF /hog1 in the supplemental material). All figure panels were derived from the same set of plates. (B) To examine Hog1 phosphorylation following exposure to stress, C. albicans RM1000+CIp20 cells were exposed to stress and extracts were prepared and subjected to Western blotting with a phospho-p38 antibody to detect phosphorylated Hog1 (P-Hog1) and with an anti-Hog1 antibody to detect total Hog1 levels (Hog1) after 10 min of exposure to 1 M NaCl and 10 min of exposure to the DPTA-NONOate carrier NaOH at 0.01 mM; then, expression was detected after exposure to 2.5 mM DPTA-NONOate, 6.0 mM DPTA-NONOate, or 5 mM NaNO 2 at the indicated times (in minutes). All figure panels were derived from the same set of Western blots. (C) Localization of Hog1-YFP in C. albicans JC63 cells exposed for 10 min to no stress (control), 5 mM H 2 O 2 , 2.5 mM DPTA-NONOate, or 5 mM NaNO 2 . Nuclei were counterstained with DAPI. Representative images of cells examined by differential interference contrast (DIC) and fluorescence microscopy (Hog1-YFP and DAPI) are shown.

    Journal: mBio

    Article Title: Redox Regulation, Rather than Stress-Induced Phosphorylation, of a Hog1 Mitogen-Activated Protein Kinase Modulates Its Nitrosative-Stress-Specific Outputs

    doi: 10.1128/mBio.02229-17

    Figure Lengend Snippet: Hog1 is required for nitrosative-stress resistance. (A) To assay stress resistance, the following C. albicans strains were spotted in 10-fold serial dilutions on plates containing no stress (control), 25 mM succinic acid (SA), 5 mM NaNO 2 , 25 mM succinic acid plus 5 mM NaNO 2 , 0.01 mM NaOH, or 2.5 mM DPTA-NONOate in 0.01 mM NaOH: RM1000+CIp20 ( HOG1/HOG1 ), Ca2226 ( HOG1/hog1 ), JC50 ( hog1/hog1 ), and JC76 ( hog1 AF /hog1 in the supplemental material). All figure panels were derived from the same set of plates. (B) To examine Hog1 phosphorylation following exposure to stress, C. albicans RM1000+CIp20 cells were exposed to stress and extracts were prepared and subjected to Western blotting with a phospho-p38 antibody to detect phosphorylated Hog1 (P-Hog1) and with an anti-Hog1 antibody to detect total Hog1 levels (Hog1) after 10 min of exposure to 1 M NaCl and 10 min of exposure to the DPTA-NONOate carrier NaOH at 0.01 mM; then, expression was detected after exposure to 2.5 mM DPTA-NONOate, 6.0 mM DPTA-NONOate, or 5 mM NaNO 2 at the indicated times (in minutes). All figure panels were derived from the same set of Western blots. (C) Localization of Hog1-YFP in C. albicans JC63 cells exposed for 10 min to no stress (control), 5 mM H 2 O 2 , 2.5 mM DPTA-NONOate, or 5 mM NaNO 2 . Nuclei were counterstained with DAPI. Representative images of cells examined by differential interference contrast (DIC) and fluorescence microscopy (Hog1-YFP and DAPI) are shown.

    Article Snippet: Phosphorylated Hog1 was detected using a phospho-p38 antibody (Thr180/Tyr182 number 9211; Cell Signalling Technology, Leiden, The Netherlands), total Hog1 was examined with an anti-Hog1 antibody (y-215, sc-9079; Santa Cruz Biotechnology, Heidelberg, Germany), and actin was analyzed with an antiactin antibody (A5060; Sigma-Aldrich, Dorset, UK).

    Techniques: Derivative Assay, Western Blot, Expressing, Fluorescence, Microscopy

    TGFβ2 exhibits higher potency in activating both canonical and non-canonical pathways in HMECs compared to TGFβ1 and TGFβ3 (A) Representative Western blot images and the corresponding bar graph of band densitometry showing increased phosphorylation and total expression of canonical transcription factor Smad2/3 in HMECs in response to 1, 2.5 and 5 ng/ml of TGFβ1, TGFβ2, and TGFβ3 for 72 hours. (B) Representative Western blot images and the corresponding bar graph of band densitometry showing increased phosphorylation of non-canonical, stress-induced p38 MAPK in HMECs in response to 1, 2.5 and 5 ng/ml of TGFβ1, TGFβ2, and TGFβ3 for 72 hours. Data are represented as mean ± SD. (n=3–5), *p

    Journal: Journal of cellular physiology

    Article Title: Isoform-Specific Effects of Transforming Growth Factor-β on Endothelial to Mesenchymal Transition

    doi: 10.1002/jcp.26801

    Figure Lengend Snippet: TGFβ2 exhibits higher potency in activating both canonical and non-canonical pathways in HMECs compared to TGFβ1 and TGFβ3 (A) Representative Western blot images and the corresponding bar graph of band densitometry showing increased phosphorylation and total expression of canonical transcription factor Smad2/3 in HMECs in response to 1, 2.5 and 5 ng/ml of TGFβ1, TGFβ2, and TGFβ3 for 72 hours. (B) Representative Western blot images and the corresponding bar graph of band densitometry showing increased phosphorylation of non-canonical, stress-induced p38 MAPK in HMECs in response to 1, 2.5 and 5 ng/ml of TGFβ1, TGFβ2, and TGFβ3 for 72 hours. Data are represented as mean ± SD. (n=3–5), *p

    Article Snippet: Antibodies used include N-cadherin (4061), VE-cadherin (2158S), phosphorylated p-38 MAPK (9211S), total p38-MAPK (9212S), phosphorylated Smad2/3 (8828S), total Smad2/3 (8685S), FoxC2 (12974S), Snail (3879S), and GAPDH (2118L) from Cell Signaling Technology (Danvers, MA), αSMA (A2547) and β-actin (A5441) from Sigma (St. Louis, MO), eNOS (610297) from BD Pharmingen (San Diego, CA), and TGFβ2 (MAB612) from R & D (Minneapolis, MN).

    Techniques: Western Blot, Expressing

    Blockage of NF-κB p65 and/or MAPK p38 attenuates B7-H3-amplified proinflammatory cytokine and chemokine production in brain tissues of S . pneumoniae -infected mice. Mice were challenged with PBS as the control, live S . pneumoniae (SP), or live S . pneumoniae plus B7-H3 (SP+B7-H3) 1 hr after mice pretreated with the MAPK p38 inhibitor SB203580, the NF-κB p65 inhibitor PDTC, or their combination (SB203580+PDTC) as described in the Methods. Brain samples were collected at 18 hrs after challenges for detecting protein levels ( B ) of TNF-α ( A ), IL-1β ( B ), IL-6 ( C ), and MCP-1 ( D ) by ELISA. Data are expressed as mean ± SD of five to six mice per time point and represent two separate experiments. ** p

    Journal: PLoS ONE

    Article Title: B7-H3 Augments Inflammatory Responses and Exacerbates Brain Damage via Amplifying NF-κB p65 and MAPK p38 Activation during Experimental Pneumococcal Meningitis

    doi: 10.1371/journal.pone.0171146

    Figure Lengend Snippet: Blockage of NF-κB p65 and/or MAPK p38 attenuates B7-H3-amplified proinflammatory cytokine and chemokine production in brain tissues of S . pneumoniae -infected mice. Mice were challenged with PBS as the control, live S . pneumoniae (SP), or live S . pneumoniae plus B7-H3 (SP+B7-H3) 1 hr after mice pretreated with the MAPK p38 inhibitor SB203580, the NF-κB p65 inhibitor PDTC, or their combination (SB203580+PDTC) as described in the Methods. Brain samples were collected at 18 hrs after challenges for detecting protein levels ( B ) of TNF-α ( A ), IL-1β ( B ), IL-6 ( C ), and MCP-1 ( D ) by ELISA. Data are expressed as mean ± SD of five to six mice per time point and represent two separate experiments. ** p

    Article Snippet: Abs that recognize NF-κB p65, phospho-p65 at Ser536, MAPK p38, and phospho-p38 at Thr180/Tyr182 were purchased from Santa Cruz Biotechnology and Cell Signaling Technology (Beverly, MA, USA), respectively.

    Techniques: Amplification, Infection, Mouse Assay, Enzyme-linked Immunosorbent Assay

    Blockage of NF-κB p65 and/or MAPK p38 attenuates B7-H3-amplified proinflammatory cytokine and chemokine mRNA expression in brain tissues of S . pneumoniae -infected mice. Mice were challenged with PBS as the control, live S . pneumoniae (SP), or live S . pneumoniae plus B7-H3 (SP+B7-H3) 1 hr after mice pretreated with the MAPK p38 inhibitor SB203580, the NF-κB p65 inhibitor PDTC, or their combination (SB203580+PDTC) as described in the Methods. Brain samples were collected at 12 hrs after challenges for detecting mRNA expression of TNF-α ( A ), IL-1β ( B ), IL-6 ( C ), and MCP-1 ( D ) by quantitative real-time PCR. Data are expressed as mean ± SD of five to six mice per time point and represent two separate experiments. ** p

    Journal: PLoS ONE

    Article Title: B7-H3 Augments Inflammatory Responses and Exacerbates Brain Damage via Amplifying NF-κB p65 and MAPK p38 Activation during Experimental Pneumococcal Meningitis

    doi: 10.1371/journal.pone.0171146

    Figure Lengend Snippet: Blockage of NF-κB p65 and/or MAPK p38 attenuates B7-H3-amplified proinflammatory cytokine and chemokine mRNA expression in brain tissues of S . pneumoniae -infected mice. Mice were challenged with PBS as the control, live S . pneumoniae (SP), or live S . pneumoniae plus B7-H3 (SP+B7-H3) 1 hr after mice pretreated with the MAPK p38 inhibitor SB203580, the NF-κB p65 inhibitor PDTC, or their combination (SB203580+PDTC) as described in the Methods. Brain samples were collected at 12 hrs after challenges for detecting mRNA expression of TNF-α ( A ), IL-1β ( B ), IL-6 ( C ), and MCP-1 ( D ) by quantitative real-time PCR. Data are expressed as mean ± SD of five to six mice per time point and represent two separate experiments. ** p

    Article Snippet: Abs that recognize NF-κB p65, phospho-p65 at Ser536, MAPK p38, and phospho-p38 at Thr180/Tyr182 were purchased from Santa Cruz Biotechnology and Cell Signaling Technology (Beverly, MA, USA), respectively.

    Techniques: Amplification, Expressing, Infection, Mouse Assay, Real-time Polymerase Chain Reaction

    Administration of B7-H3 up-regulates phosphorylation of NF-κB p65 and MAPK p38 in brain tissues of S . pneumoniae -infected mice. Mice were challenged with PBS as the control, recombinant mouse B7-H3, live S . pneumoniae (SP), or live S . pneumoniae plus B7-H3 (SP+B7-H3) via intracerebral ventricular injection as described in the Methods. Brain samples were collected at the indicated time points after challenges. Phosphorylated p65 at Ser536 ( A ), total p65 ( A ), phosphorylated p38 at Thr180/Tyr182 ( B ), and total p38 ( B ) was detected by Western blot analysis. Data shown represent one experiment from a total of four separate experiments. Density ratios of p-p65/p65 (n = 4) ( C ) and p-p38/p38 (n = 4) ( D ) were quantified by densitometry analysis. ** p

    Journal: PLoS ONE

    Article Title: B7-H3 Augments Inflammatory Responses and Exacerbates Brain Damage via Amplifying NF-κB p65 and MAPK p38 Activation during Experimental Pneumococcal Meningitis

    doi: 10.1371/journal.pone.0171146

    Figure Lengend Snippet: Administration of B7-H3 up-regulates phosphorylation of NF-κB p65 and MAPK p38 in brain tissues of S . pneumoniae -infected mice. Mice were challenged with PBS as the control, recombinant mouse B7-H3, live S . pneumoniae (SP), or live S . pneumoniae plus B7-H3 (SP+B7-H3) via intracerebral ventricular injection as described in the Methods. Brain samples were collected at the indicated time points after challenges. Phosphorylated p65 at Ser536 ( A ), total p65 ( A ), phosphorylated p38 at Thr180/Tyr182 ( B ), and total p38 ( B ) was detected by Western blot analysis. Data shown represent one experiment from a total of four separate experiments. Density ratios of p-p65/p65 (n = 4) ( C ) and p-p38/p38 (n = 4) ( D ) were quantified by densitometry analysis. ** p

    Article Snippet: Abs that recognize NF-κB p65, phospho-p65 at Ser536, MAPK p38, and phospho-p38 at Thr180/Tyr182 were purchased from Santa Cruz Biotechnology and Cell Signaling Technology (Beverly, MA, USA), respectively.

    Techniques: Infection, Mouse Assay, Recombinant, Injection, Western Blot

    Blockage of NF-κB p65 and/or MAPK p38 ameliorates B7-H3-exacerbated disruption of BBB integrity and severity of disease status in S . pneumoniae -infected mice. Mice were challenged with PBS as the control, live S . pneumoniae (SP), or live S . pneumoniae plus B7-H3 (SP+B7-H3) 1 hr after mice pretreated with the MAPK p38 inhibitor SB203580, the NF-κB p65 inhibitor PDTC, or their combination (SB203580+PDTC) as described in the Methods. Brain samples were collected at 30 hrs after challenges for detecting albumin ( A ) and IgG ( B ) levels using ELISA. The severity of disease status as represented by spontaneous motor activity ( C ) and body weight loss ( D ) was examined 30 hrs after challenges. Data are expressed as mean ± SD of five to six mice per time point and represent two separate experiments. ** p

    Journal: PLoS ONE

    Article Title: B7-H3 Augments Inflammatory Responses and Exacerbates Brain Damage via Amplifying NF-κB p65 and MAPK p38 Activation during Experimental Pneumococcal Meningitis

    doi: 10.1371/journal.pone.0171146

    Figure Lengend Snippet: Blockage of NF-κB p65 and/or MAPK p38 ameliorates B7-H3-exacerbated disruption of BBB integrity and severity of disease status in S . pneumoniae -infected mice. Mice were challenged with PBS as the control, live S . pneumoniae (SP), or live S . pneumoniae plus B7-H3 (SP+B7-H3) 1 hr after mice pretreated with the MAPK p38 inhibitor SB203580, the NF-κB p65 inhibitor PDTC, or their combination (SB203580+PDTC) as described in the Methods. Brain samples were collected at 30 hrs after challenges for detecting albumin ( A ) and IgG ( B ) levels using ELISA. The severity of disease status as represented by spontaneous motor activity ( C ) and body weight loss ( D ) was examined 30 hrs after challenges. Data are expressed as mean ± SD of five to six mice per time point and represent two separate experiments. ** p

    Article Snippet: Abs that recognize NF-κB p65, phospho-p65 at Ser536, MAPK p38, and phospho-p38 at Thr180/Tyr182 were purchased from Santa Cruz Biotechnology and Cell Signaling Technology (Beverly, MA, USA), respectively.

    Techniques: Infection, Mouse Assay, Enzyme-linked Immunosorbent Assay, Activity Assay

    (A) Some residual activity is associated with Irak D358N which is sufficient to induce some spreading of macrophages. Macrophages were microinjected with FITC-dextran (control) or cDNAs encoding myc-tagged Irak, Irak D358N, or Irak N. Cells were then fixed, stained with anti-myc antibody and rhodamine-phalloidin, and assayed for spreading. (B) Overexpression of Irak D358N induces some p38 activation. COS-1 cells were transfected with cDNA encoding for p38α along with control DNA (−), a cDNA construct encoding for myc-tagged Irak, or with increasing amounts of cDNA encoding for myc-tagged Irak D358N. Whole cell extracts were prepared and subjected to SDS-PAGE and Western analysis using anti-phospho-p38 (top panel), anti-p38 (middle panel), and anti-myc (bottom panel) antibodies.

    Journal: Molecular and Cellular Biology

    Article Title: Lipopolysaccharide-Induced Activation of ?2-Integrin Function in Macrophages Requires Irak Kinase Activity, p38 Mitogen- Activated Protein Kinase, and the Rap1 GTPase

    doi: 10.1128/MCB.21.2.438-448.2001

    Figure Lengend Snippet: (A) Some residual activity is associated with Irak D358N which is sufficient to induce some spreading of macrophages. Macrophages were microinjected with FITC-dextran (control) or cDNAs encoding myc-tagged Irak, Irak D358N, or Irak N. Cells were then fixed, stained with anti-myc antibody and rhodamine-phalloidin, and assayed for spreading. (B) Overexpression of Irak D358N induces some p38 activation. COS-1 cells were transfected with cDNA encoding for p38α along with control DNA (−), a cDNA construct encoding for myc-tagged Irak, or with increasing amounts of cDNA encoding for myc-tagged Irak D358N. Whole cell extracts were prepared and subjected to SDS-PAGE and Western analysis using anti-phospho-p38 (top panel), anti-p38 (middle panel), and anti-myc (bottom panel) antibodies.

    Article Snippet: Polyclonal antibodies to p38 and to dually phosphorylated p38 (Thr180, Tyr182) were purchased from Santa Cruz and New England Biolabs, respectively.

    Techniques: Activity Assay, Staining, Over Expression, Activation Assay, Transfection, Construct, SDS Page, Western Blot

    Model of LPS-induced signaling pathways leading to activation of transcription and spreading. LPS-induced spreading is mediated by a linear pathway, comprising MyD88, Irak, p38, Rap1, and β2-integrins. See Discussion for further details.

    Journal: Molecular and Cellular Biology

    Article Title: Lipopolysaccharide-Induced Activation of ?2-Integrin Function in Macrophages Requires Irak Kinase Activity, p38 Mitogen- Activated Protein Kinase, and the Rap1 GTPase

    doi: 10.1128/MCB.21.2.438-448.2001

    Figure Lengend Snippet: Model of LPS-induced signaling pathways leading to activation of transcription and spreading. LPS-induced spreading is mediated by a linear pathway, comprising MyD88, Irak, p38, Rap1, and β2-integrins. See Discussion for further details.

    Article Snippet: Polyclonal antibodies to p38 and to dually phosphorylated p38 (Thr180, Tyr182) were purchased from Santa Cruz and New England Biolabs, respectively.

    Techniques: Activation Assay

    (A) LPS-induced spreading requires Irak and MyD88; Irak-induced spreading is dependent on p38 and Rap1. Shown here is the induction of macrophage spreading by Irak and MyD88 constructs in the presence or absence of LPS, SB202190, or TcdB-1470. Macrophages were microinjected with FITC-dextran (−) or cDNAs encoding for myc-tagged Irak wild-type and mutant constructs or AU1-tagged MyD88▵. Cells were returned to the incubator for expression of the constructs and then incubated as indicated either with LPS (1 μg/ml) for 10 min, SB202190 (1 μM) for 20 min, or TcdB-1470 (10 pg/ml) for 2 h. Cells were then fixed, stained with anti-myc or anti-AU1 antibodies and rhodamine-phalloidin, and assayed for spreading. (B) Irak induces spreading in J774.A1 macrophages. Macrophages were microinjected with FITC-dextran (control) or a cDNA construct encoding for myc-tagged Irak. Cells were returned to the incubator for expression of the construct, fixed and stained with anti-myc and rhodamine-phalloidin, and assayed for spreading.

    Journal: Molecular and Cellular Biology

    Article Title: Lipopolysaccharide-Induced Activation of ?2-Integrin Function in Macrophages Requires Irak Kinase Activity, p38 Mitogen- Activated Protein Kinase, and the Rap1 GTPase

    doi: 10.1128/MCB.21.2.438-448.2001

    Figure Lengend Snippet: (A) LPS-induced spreading requires Irak and MyD88; Irak-induced spreading is dependent on p38 and Rap1. Shown here is the induction of macrophage spreading by Irak and MyD88 constructs in the presence or absence of LPS, SB202190, or TcdB-1470. Macrophages were microinjected with FITC-dextran (−) or cDNAs encoding for myc-tagged Irak wild-type and mutant constructs or AU1-tagged MyD88▵. Cells were returned to the incubator for expression of the constructs and then incubated as indicated either with LPS (1 μg/ml) for 10 min, SB202190 (1 μM) for 20 min, or TcdB-1470 (10 pg/ml) for 2 h. Cells were then fixed, stained with anti-myc or anti-AU1 antibodies and rhodamine-phalloidin, and assayed for spreading. (B) Irak induces spreading in J774.A1 macrophages. Macrophages were microinjected with FITC-dextran (control) or a cDNA construct encoding for myc-tagged Irak. Cells were returned to the incubator for expression of the construct, fixed and stained with anti-myc and rhodamine-phalloidin, and assayed for spreading.

    Article Snippet: Polyclonal antibodies to p38 and to dually phosphorylated p38 (Thr180, Tyr182) were purchased from Santa Cruz and New England Biolabs, respectively.

    Techniques: Construct, Mutagenesis, Expressing, Incubation, Staining

    p38 MAP kinase is required for LPS-induced spreading of J774.A1 macrophages. (A and B) SB202190 blocks LPS-induced spreading of macrophages. Macrophages were treated with or without SB202190 (A, 1 μM; B, 0.1 to 10 μM) for 20 min at 37°C, incubated with or without LPS (1 μg/ml) for 10 min, fixed, stained with rhodamine-phalloidin, and then assayed for spreading. (C) Expression of dominant-negative p38 inhibits LPS-induced spreading. Macrophages were microinjected with FITC-dextran (−) or a cDNA construct encoding for HA-tagged dnp38, returned to the incubator for expression of the construct, and then treated with or without LPS (1 μg/ml) for 10 min. Cells were fixed and stained with anti-HA antibody and rhodamine-phalloidin, and the percentage of injected/expressing spread cells was determined. (D) The time course of LPS-induced spreading correlates with the time course of p38 activation. (Top and middle panels) Macrophages were incubated with LPS (1 μg/ml) for the indicated times and then lysed. Whole cell extracts were prepared and subjected to SDS-PAGE and Western analysis using anti-p38 (middle) and anti-phospho-p38 (top) antibodies. (Bottom panel) Macrophages were incubated with LPS (1 μg/ml) for the indicated times, fixed, and stained with rhodamine-phalloidin, and the percentage of spread cells was determined.

    Journal: Molecular and Cellular Biology

    Article Title: Lipopolysaccharide-Induced Activation of ?2-Integrin Function in Macrophages Requires Irak Kinase Activity, p38 Mitogen- Activated Protein Kinase, and the Rap1 GTPase

    doi: 10.1128/MCB.21.2.438-448.2001

    Figure Lengend Snippet: p38 MAP kinase is required for LPS-induced spreading of J774.A1 macrophages. (A and B) SB202190 blocks LPS-induced spreading of macrophages. Macrophages were treated with or without SB202190 (A, 1 μM; B, 0.1 to 10 μM) for 20 min at 37°C, incubated with or without LPS (1 μg/ml) for 10 min, fixed, stained with rhodamine-phalloidin, and then assayed for spreading. (C) Expression of dominant-negative p38 inhibits LPS-induced spreading. Macrophages were microinjected with FITC-dextran (−) or a cDNA construct encoding for HA-tagged dnp38, returned to the incubator for expression of the construct, and then treated with or without LPS (1 μg/ml) for 10 min. Cells were fixed and stained with anti-HA antibody and rhodamine-phalloidin, and the percentage of injected/expressing spread cells was determined. (D) The time course of LPS-induced spreading correlates with the time course of p38 activation. (Top and middle panels) Macrophages were incubated with LPS (1 μg/ml) for the indicated times and then lysed. Whole cell extracts were prepared and subjected to SDS-PAGE and Western analysis using anti-p38 (middle) and anti-phospho-p38 (top) antibodies. (Bottom panel) Macrophages were incubated with LPS (1 μg/ml) for the indicated times, fixed, and stained with rhodamine-phalloidin, and the percentage of spread cells was determined.

    Article Snippet: Polyclonal antibodies to p38 and to dually phosphorylated p38 (Thr180, Tyr182) were purchased from Santa Cruz and New England Biolabs, respectively.

    Techniques: Incubation, Staining, Expressing, Dominant Negative Mutation, Construct, Injection, Activation Assay, SDS Page, Western Blot

    (A and B) Irak's kinase activity is required for activation of p38 in J774.A1 macrophages. Macrophages were microinjected with Texas red-dextran (control) or cDNAs encoding myc-tagged Irak, Irak D358N, or Irak N. Cells were fixed and stained with anti-myc (C, E, and G) or anti-phospho-p38 (B, D, F, and H) antibodies. The percentage of cells with activated p38 was determined. (C) Irak's kinase activity is required for activation of p38 in COS-1 cells. cDNAs encoding myc-tagged Irak, Irak D358N, or Irak N and control DNA (−) together with cDNA encoding for p38α were transfected into COS-1 cells. Whole cell extracts were prepared and subjected to SDS-PAGE and Western analysis using anti-phospho-p38 (top panel), anti-p38 (middle panel), and anti-myc (bottom panel) antibodies. The band above Irak N, which is visible in all four lanes, is due to cross-reactivity with the secondary antibody.

    Journal: Molecular and Cellular Biology

    Article Title: Lipopolysaccharide-Induced Activation of ?2-Integrin Function in Macrophages Requires Irak Kinase Activity, p38 Mitogen- Activated Protein Kinase, and the Rap1 GTPase

    doi: 10.1128/MCB.21.2.438-448.2001

    Figure Lengend Snippet: (A and B) Irak's kinase activity is required for activation of p38 in J774.A1 macrophages. Macrophages were microinjected with Texas red-dextran (control) or cDNAs encoding myc-tagged Irak, Irak D358N, or Irak N. Cells were fixed and stained with anti-myc (C, E, and G) or anti-phospho-p38 (B, D, F, and H) antibodies. The percentage of cells with activated p38 was determined. (C) Irak's kinase activity is required for activation of p38 in COS-1 cells. cDNAs encoding myc-tagged Irak, Irak D358N, or Irak N and control DNA (−) together with cDNA encoding for p38α were transfected into COS-1 cells. Whole cell extracts were prepared and subjected to SDS-PAGE and Western analysis using anti-phospho-p38 (top panel), anti-p38 (middle panel), and anti-myc (bottom panel) antibodies. The band above Irak N, which is visible in all four lanes, is due to cross-reactivity with the secondary antibody.

    Article Snippet: Polyclonal antibodies to p38 and to dually phosphorylated p38 (Thr180, Tyr182) were purchased from Santa Cruz and New England Biolabs, respectively.

    Techniques: Activity Assay, Activation Assay, Staining, Transfection, SDS Page, Western Blot

    Rap1 is downstream of p38. (A and B) TcdB1470 does not inhibit LPS-induced p38 activation in J774.A1 macrophages. Macrophages were treated with or without TcdB-1470 (10 pg/ml) for 2 h at 37°C and then incubated with or without LPS (1 μg/ml) for 10 min. Cells were fixed and stained with anti-phospho-p38 antibody (A), or cells were lysed and whole cell extracts were subjected to SDS-PAGE and Western analysis using the anti-phospho-p38 and anti-p38 antibodies (B). (C and D) SB202190 inhibits LPS-induced Rap1 activation in macrophages. Cells were incubated with or without SB202190 (5 μM) for 20 min at 37°C, incubated with or without LPS (0.1 μg/ml) for 15 min, and lysed. Extracts were incubated with 20 μg of Gst-RalGDS-RBD precoupled to gluthathione beads to isolate GTP-bound (activated) Rap1. Levels of activated Rap1 were monitored by Western analysis with anti-Rap1 antibody (upper panel). Levels of Rap1 in total lysates are shown in the lower panel. (D) Western blots of five individual experiments were scanned and quantified. Rap1 GTP levels were normalized to total Rap1 levels, and the activation of Rap1 by LPS in the presence or absence of SB202190 was calculated as fold activation of Rap1 compared to non-LPS-treated cells.

    Journal: Molecular and Cellular Biology

    Article Title: Lipopolysaccharide-Induced Activation of ?2-Integrin Function in Macrophages Requires Irak Kinase Activity, p38 Mitogen- Activated Protein Kinase, and the Rap1 GTPase

    doi: 10.1128/MCB.21.2.438-448.2001

    Figure Lengend Snippet: Rap1 is downstream of p38. (A and B) TcdB1470 does not inhibit LPS-induced p38 activation in J774.A1 macrophages. Macrophages were treated with or without TcdB-1470 (10 pg/ml) for 2 h at 37°C and then incubated with or without LPS (1 μg/ml) for 10 min. Cells were fixed and stained with anti-phospho-p38 antibody (A), or cells were lysed and whole cell extracts were subjected to SDS-PAGE and Western analysis using the anti-phospho-p38 and anti-p38 antibodies (B). (C and D) SB202190 inhibits LPS-induced Rap1 activation in macrophages. Cells were incubated with or without SB202190 (5 μM) for 20 min at 37°C, incubated with or without LPS (0.1 μg/ml) for 15 min, and lysed. Extracts were incubated with 20 μg of Gst-RalGDS-RBD precoupled to gluthathione beads to isolate GTP-bound (activated) Rap1. Levels of activated Rap1 were monitored by Western analysis with anti-Rap1 antibody (upper panel). Levels of Rap1 in total lysates are shown in the lower panel. (D) Western blots of five individual experiments were scanned and quantified. Rap1 GTP levels were normalized to total Rap1 levels, and the activation of Rap1 by LPS in the presence or absence of SB202190 was calculated as fold activation of Rap1 compared to non-LPS-treated cells.

    Article Snippet: Polyclonal antibodies to p38 and to dually phosphorylated p38 (Thr180, Tyr182) were purchased from Santa Cruz and New England Biolabs, respectively.

    Techniques: Activation Assay, Incubation, Staining, SDS Page, Western Blot

    Effect of p38 kinase stimulated by DMP1 on mineralized nodule formation. MC3T3-E1 cells were grown in mineralization media in the presence of DMP1 or SB203580 and DMP1 for 7, 14, and 21 days. Mineralized nodules were assayed with von Kossa staining. DMP1 stimulated the formation of mineralized nodules at 14 and 21 days, although SB203580 inhibitor suppressed nodule formation.

    Journal: The Journal of Biological Chemistry

    Article Title: Calcium-mediated Stress Kinase Activation by DMP1 Promotes Osteoblast Differentiation *

    doi: 10.1074/jbc.M110.145607

    Figure Lengend Snippet: Effect of p38 kinase stimulated by DMP1 on mineralized nodule formation. MC3T3-E1 cells were grown in mineralization media in the presence of DMP1 or SB203580 and DMP1 for 7, 14, and 21 days. Mineralized nodules were assayed with von Kossa staining. DMP1 stimulated the formation of mineralized nodules at 14 and 21 days, although SB203580 inhibitor suppressed nodule formation.

    Article Snippet: The cells were then washed with PBS and blocked with 5% BSA in PBS for 1 h. After blocking, the cells were then incubated overnight with anti-phospho-p38 (1:100) (Santa Cruz Biotechnology) or with anti-phospho-HSP27 (1:100) (Cell Signaling) and followed by a 1-h incubation with a fluorescein-conjugated goat anti-rabbit IgG (Sigma).

    Techniques: Staining

    DMP1 stimulates phosphorylation of p38 MAPK in MC3T3-E1 and C3H10T1/2 cells and primary calvarial cells. Cells in basal medium were untreated ( control ) or treated with rDMP1 (250 ng/ml) for 15, 30, 45, and 60 min and 1.5 and 2 h. Cell lysates were harvested and subjected to SDS-PAGE, and Western blot analysis was performed with phospho-p38 antibody. The blots were stripped and probed for total p38. Stimulation by DMP1 phosphorylates p38 MAPK in MC3T3-E1 cells ( A ), C3H10T1/2 cells ( B ), and primary calvarial cells ( C ). The graphs show the quantification of the percentage levels of phosphorylated p38. MC3T3-E1 cells stimulated with native DMP1 showed activation of p38 MAPK ( D ).

    Journal: The Journal of Biological Chemistry

    Article Title: Calcium-mediated Stress Kinase Activation by DMP1 Promotes Osteoblast Differentiation *

    doi: 10.1074/jbc.M110.145607

    Figure Lengend Snippet: DMP1 stimulates phosphorylation of p38 MAPK in MC3T3-E1 and C3H10T1/2 cells and primary calvarial cells. Cells in basal medium were untreated ( control ) or treated with rDMP1 (250 ng/ml) for 15, 30, 45, and 60 min and 1.5 and 2 h. Cell lysates were harvested and subjected to SDS-PAGE, and Western blot analysis was performed with phospho-p38 antibody. The blots were stripped and probed for total p38. Stimulation by DMP1 phosphorylates p38 MAPK in MC3T3-E1 cells ( A ), C3H10T1/2 cells ( B ), and primary calvarial cells ( C ). The graphs show the quantification of the percentage levels of phosphorylated p38. MC3T3-E1 cells stimulated with native DMP1 showed activation of p38 MAPK ( D ).

    Article Snippet: The cells were then washed with PBS and blocked with 5% BSA in PBS for 1 h. After blocking, the cells were then incubated overnight with anti-phospho-p38 (1:100) (Santa Cruz Biotechnology) or with anti-phospho-HSP27 (1:100) (Cell Signaling) and followed by a 1-h incubation with a fluorescein-conjugated goat anti-rabbit IgG (Sigma).

    Techniques: SDS Page, Western Blot, Activation Assay

    Effect of DMP1 on osteogenic gene expression and their abrogation in the presence of p38 inhibitor SB203580 and PKC inhibitor calphostin. MC3T3-E1 cells were either left untreated (control) or starved for 24 h prior to stimulation with 250 ng/ml rDMP1 peptide or with SB203580 and rDMP1 or with calphostin C and rDMP in basal medium for 4 and 24 h. Total RNA was isolated and subjected to real time PCR and analyzed for the expression of Runx2 ( A ) and osteocalcin ( B ). Similar experiments were performed with C3H10T1/2 cells (Runx2 ( C ) and osteocalcin ( D )). Stimulation with DMP1 significantly up-regulated the expression levels of Runx2 and osteocalcin. Treatment with SB203580, a specific inhibitor for p38 MAPK activation or calphostin, down-regulated gene expression. Note basal levels of gene expression were not affected by the addition of SB203580 alone. These results were normalized with the loading control GAPDH. Experiments were done in triplicate.

    Journal: The Journal of Biological Chemistry

    Article Title: Calcium-mediated Stress Kinase Activation by DMP1 Promotes Osteoblast Differentiation *

    doi: 10.1074/jbc.M110.145607

    Figure Lengend Snippet: Effect of DMP1 on osteogenic gene expression and their abrogation in the presence of p38 inhibitor SB203580 and PKC inhibitor calphostin. MC3T3-E1 cells were either left untreated (control) or starved for 24 h prior to stimulation with 250 ng/ml rDMP1 peptide or with SB203580 and rDMP1 or with calphostin C and rDMP in basal medium for 4 and 24 h. Total RNA was isolated and subjected to real time PCR and analyzed for the expression of Runx2 ( A ) and osteocalcin ( B ). Similar experiments were performed with C3H10T1/2 cells (Runx2 ( C ) and osteocalcin ( D )). Stimulation with DMP1 significantly up-regulated the expression levels of Runx2 and osteocalcin. Treatment with SB203580, a specific inhibitor for p38 MAPK activation or calphostin, down-regulated gene expression. Note basal levels of gene expression were not affected by the addition of SB203580 alone. These results were normalized with the loading control GAPDH. Experiments were done in triplicate.

    Article Snippet: The cells were then washed with PBS and blocked with 5% BSA in PBS for 1 h. After blocking, the cells were then incubated overnight with anti-phospho-p38 (1:100) (Santa Cruz Biotechnology) or with anti-phospho-HSP27 (1:100) (Cell Signaling) and followed by a 1-h incubation with a fluorescein-conjugated goat anti-rabbit IgG (Sigma).

    Techniques: Expressing, Isolation, Real-time Polymerase Chain Reaction, Activation Assay

    DMP1 stimulates the nuclear translocation of phospho-p38. A , immunofluorescence analysis showing localization of phospho-p38 ( panel 2 ) in MC3T3-E1 cells stimulated with rDMP1 at the indicated time points. DNA was stained with DAPI ( panel 1 ). Note nuclear localization of phospho-p38 in the nucleus as early as 30 min and overlap between the two images is depicted in panel 3 . The scale bar indicates 20 μm. B , total proteins were isolated from the nuclear and cytoplasmic compartment of MC3T3-E1 cells stimulated with DMP1 at the indicated time point. Western blot analysis was performed with anti-phospho-p38 antibody.

    Journal: The Journal of Biological Chemistry

    Article Title: Calcium-mediated Stress Kinase Activation by DMP1 Promotes Osteoblast Differentiation *

    doi: 10.1074/jbc.M110.145607

    Figure Lengend Snippet: DMP1 stimulates the nuclear translocation of phospho-p38. A , immunofluorescence analysis showing localization of phospho-p38 ( panel 2 ) in MC3T3-E1 cells stimulated with rDMP1 at the indicated time points. DNA was stained with DAPI ( panel 1 ). Note nuclear localization of phospho-p38 in the nucleus as early as 30 min and overlap between the two images is depicted in panel 3 . The scale bar indicates 20 μm. B , total proteins were isolated from the nuclear and cytoplasmic compartment of MC3T3-E1 cells stimulated with DMP1 at the indicated time point. Western blot analysis was performed with anti-phospho-p38 antibody.

    Article Snippet: The cells were then washed with PBS and blocked with 5% BSA in PBS for 1 h. After blocking, the cells were then incubated overnight with anti-phospho-p38 (1:100) (Santa Cruz Biotechnology) or with anti-phospho-HSP27 (1:100) (Cell Signaling) and followed by a 1-h incubation with a fluorescein-conjugated goat anti-rabbit IgG (Sigma).

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

    Effect of DMP1 on the expression of Gα q11 and a hypothetical model. A , total proteins were extracted from DMP1-stimulated MC3T3-E1 at the indicated time points. Western blot analysis was performed with anti-Gα q11 antibody. Tubulin was used as the loading control. B , hypothetical model depicting the depletion of ER Ca 2+ stores upon DMP1 endocytosis and the subsequent activation of p38 MAPK and downstream gene transcription.

    Journal: The Journal of Biological Chemistry

    Article Title: Calcium-mediated Stress Kinase Activation by DMP1 Promotes Osteoblast Differentiation *

    doi: 10.1074/jbc.M110.145607

    Figure Lengend Snippet: Effect of DMP1 on the expression of Gα q11 and a hypothetical model. A , total proteins were extracted from DMP1-stimulated MC3T3-E1 at the indicated time points. Western blot analysis was performed with anti-Gα q11 antibody. Tubulin was used as the loading control. B , hypothetical model depicting the depletion of ER Ca 2+ stores upon DMP1 endocytosis and the subsequent activation of p38 MAPK and downstream gene transcription.

    Article Snippet: The cells were then washed with PBS and blocked with 5% BSA in PBS for 1 h. After blocking, the cells were then incubated overnight with anti-phospho-p38 (1:100) (Santa Cruz Biotechnology) or with anti-phospho-HSP27 (1:100) (Cell Signaling) and followed by a 1-h incubation with a fluorescein-conjugated goat anti-rabbit IgG (Sigma).

    Techniques: Expressing, Western Blot, Activation Assay

    DMP1 stimulation activates MAPKAPK2 and HSP27 that are downstream targets of the p38 MAPK signaling pathway. Confluent adherent MC3T3-E1 cells were treated without (control, C ) or with DMP1, and Western blot analysis was performed with anti-phospho-MAPKAPK2 and total MAPKAPK2. A , phosphorylation of MAPKAPK2 was assessed following DMP1 stimulation. Densitometric quantification of the blots was performed by assessing tubulin and total MAPKAPK2. B , phosphorylation of MAPKAPK2 was assessed following treatment with SB203580 and then stimulated by DMP1. C , Western blot was also performed with anti-phospho-HSP27 antibody after stimulating cells without (control) or with DMP1. Equal amounts of proteins were loaded as assessed by tubulin. D , confocal microscopy images showing nuclear localization of HSP27 in DMP1-stimulated cells. The scale bar indicates 20 μm. MC3T3-E1 cells were transfected with MAPKAPK2 wild type ( WT ), dominant negative kinase-inactive mutant ( DN ), or constitutively active mutant ( CA ) or with empty vector ( EV ). 48 h after transfection, cells were changed to serum-free media, and 24 h later cells were treated with or without DMP1 for 1 h, and phospho-HSP27 ( E ), phospho-MAPKAPK2 ( F ), and tubulin were detected by Western blot analysis.

    Journal: The Journal of Biological Chemistry

    Article Title: Calcium-mediated Stress Kinase Activation by DMP1 Promotes Osteoblast Differentiation *

    doi: 10.1074/jbc.M110.145607

    Figure Lengend Snippet: DMP1 stimulation activates MAPKAPK2 and HSP27 that are downstream targets of the p38 MAPK signaling pathway. Confluent adherent MC3T3-E1 cells were treated without (control, C ) or with DMP1, and Western blot analysis was performed with anti-phospho-MAPKAPK2 and total MAPKAPK2. A , phosphorylation of MAPKAPK2 was assessed following DMP1 stimulation. Densitometric quantification of the blots was performed by assessing tubulin and total MAPKAPK2. B , phosphorylation of MAPKAPK2 was assessed following treatment with SB203580 and then stimulated by DMP1. C , Western blot was also performed with anti-phospho-HSP27 antibody after stimulating cells without (control) or with DMP1. Equal amounts of proteins were loaded as assessed by tubulin. D , confocal microscopy images showing nuclear localization of HSP27 in DMP1-stimulated cells. The scale bar indicates 20 μm. MC3T3-E1 cells were transfected with MAPKAPK2 wild type ( WT ), dominant negative kinase-inactive mutant ( DN ), or constitutively active mutant ( CA ) or with empty vector ( EV ). 48 h after transfection, cells were changed to serum-free media, and 24 h later cells were treated with or without DMP1 for 1 h, and phospho-HSP27 ( E ), phospho-MAPKAPK2 ( F ), and tubulin were detected by Western blot analysis.

    Article Snippet: The cells were then washed with PBS and blocked with 5% BSA in PBS for 1 h. After blocking, the cells were then incubated overnight with anti-phospho-p38 (1:100) (Santa Cruz Biotechnology) or with anti-phospho-HSP27 (1:100) (Cell Signaling) and followed by a 1-h incubation with a fluorescein-conjugated goat anti-rabbit IgG (Sigma).

    Techniques: Western Blot, Confocal Microscopy, Transfection, Dominant Negative Mutation, Mutagenesis, Plasmid Preparation

    Effect of p38 kinase stimulated by DMP1 on terminal differentiation of osteoblast. DMP1 enhances terminal differentiation of osteoblast in MC3T3-E1 ( B ) and C3H10T1/2 cells ( C and D ). Cells were treated with DMP1 for 7, 14, and 21 days in the presence of mineralization media and with or without SB203580. Relative amounts of mRNA for Runx2 ( A and C ) and osteocalcin ( B and D ) were determined using real time PCR. Values are normalized to the levels of GAPDH mRNA.

    Journal: The Journal of Biological Chemistry

    Article Title: Calcium-mediated Stress Kinase Activation by DMP1 Promotes Osteoblast Differentiation *

    doi: 10.1074/jbc.M110.145607

    Figure Lengend Snippet: Effect of p38 kinase stimulated by DMP1 on terminal differentiation of osteoblast. DMP1 enhances terminal differentiation of osteoblast in MC3T3-E1 ( B ) and C3H10T1/2 cells ( C and D ). Cells were treated with DMP1 for 7, 14, and 21 days in the presence of mineralization media and with or without SB203580. Relative amounts of mRNA for Runx2 ( A and C ) and osteocalcin ( B and D ) were determined using real time PCR. Values are normalized to the levels of GAPDH mRNA.

    Article Snippet: The cells were then washed with PBS and blocked with 5% BSA in PBS for 1 h. After blocking, the cells were then incubated overnight with anti-phospho-p38 (1:100) (Santa Cruz Biotechnology) or with anti-phospho-HSP27 (1:100) (Cell Signaling) and followed by a 1-h incubation with a fluorescein-conjugated goat anti-rabbit IgG (Sigma).

    Techniques: Real-time Polymerase Chain Reaction

    Apoptosis and autophagy rates in Ti, Hy (400 µg/ml)+Ti, TWEAK+Ti, TWEAK+Hy+Ti, Anisomycin+Ti and Anisomycin+Hy+Ti groups are determined using flow cytometry. (A) TWEAK overexpression and p38 MAPK activation by anisomycin were able to elevate the apoptosis rate in Ti particle-injured cells, even under Hy pretreatment. (B) TWEAK overexpression and p38 MAPK activation by anisomycin may enhance the autophagy rate in Ti particle-injured cells, even under pretreatment with Hy condition. Black, control; green, Ti; pink, Hy+Ti; light blue, TWEAK+Ti; yellow, TWEAK+Hy+Ti; blue, Anisomycin+Ti; orange, Anisomycin+Hy+Ti. Data are presented as the mean ± standard deviation. n=3. *P

    Journal: Molecular Medicine Reports

    Article Title: Hyperoside decreases the apoptosis and autophagy rates of osteoblast MC3T3-E1 cells by regulating TNF-like weak inducer of apoptosis and the p38mitogen activated protein kinase pathway

    doi: 10.3892/mmr.2018.9622

    Figure Lengend Snippet: Apoptosis and autophagy rates in Ti, Hy (400 µg/ml)+Ti, TWEAK+Ti, TWEAK+Hy+Ti, Anisomycin+Ti and Anisomycin+Hy+Ti groups are determined using flow cytometry. (A) TWEAK overexpression and p38 MAPK activation by anisomycin were able to elevate the apoptosis rate in Ti particle-injured cells, even under Hy pretreatment. (B) TWEAK overexpression and p38 MAPK activation by anisomycin may enhance the autophagy rate in Ti particle-injured cells, even under pretreatment with Hy condition. Black, control; green, Ti; pink, Hy+Ti; light blue, TWEAK+Ti; yellow, TWEAK+Hy+Ti; blue, Anisomycin+Ti; orange, Anisomycin+Hy+Ti. Data are presented as the mean ± standard deviation. n=3. *P

    Article Snippet: Membranes were incubated with the following primary specific antibodies at 4°C for 6 h and subsequently at room temperature for 4 h: Anti-caspase-3 antibody (1:500; cat. no. ab13847), anti-Bax antibody (1:1,000; cat. no. ab32503), anti-Bcl-2 antibody (1:1,000; cat. no. ab692), anti-p53 antibody (1:1,000; cat. no. ab26), anti-Beclin1 antibody (1:1,000; cat. no. ab62557), anti-LC3B antibody (1:1,000; cat. no. ab48394), anti-TWEAK antibody (1:1,000; cat. no. ab37170), anti-p38 (phospho T180+Y182) antibody (1:1,000; cat. no. ab45381), anti-p38 antibody (1:1,000; cat. no. ab31828), and anti-GAPDH antibody (1:2,000; cat. no. ab8245; all Abcam).

    Techniques: Flow Cytometry, Cytometry, Over Expression, Activation Assay, Standard Deviation

    Protein expression levels of TWEAK and the p38MAPK pathway are analyzed by western blotting in the control group, Ti (1 mg/ml) group, Hy-1 (200 µg/ml)+Ti group and Hy-2 (400 µg/ml)+Ti group. (A) Pretreatment with Hy inhibited the activation of TWEAK in MC3T3-E1 cells in Ti particle-induced injury. (B) Pretreatment with Hy inhibited the phosphorylation of p38 MAPK in MC3T3-E1 cells in Ti particle-induced injury. Data are presented as the mean ± standard deviation. n=3. **P

    Journal: Molecular Medicine Reports

    Article Title: Hyperoside decreases the apoptosis and autophagy rates of osteoblast MC3T3-E1 cells by regulating TNF-like weak inducer of apoptosis and the p38mitogen activated protein kinase pathway

    doi: 10.3892/mmr.2018.9622

    Figure Lengend Snippet: Protein expression levels of TWEAK and the p38MAPK pathway are analyzed by western blotting in the control group, Ti (1 mg/ml) group, Hy-1 (200 µg/ml)+Ti group and Hy-2 (400 µg/ml)+Ti group. (A) Pretreatment with Hy inhibited the activation of TWEAK in MC3T3-E1 cells in Ti particle-induced injury. (B) Pretreatment with Hy inhibited the phosphorylation of p38 MAPK in MC3T3-E1 cells in Ti particle-induced injury. Data are presented as the mean ± standard deviation. n=3. **P

    Article Snippet: Membranes were incubated with the following primary specific antibodies at 4°C for 6 h and subsequently at room temperature for 4 h: Anti-caspase-3 antibody (1:500; cat. no. ab13847), anti-Bax antibody (1:1,000; cat. no. ab32503), anti-Bcl-2 antibody (1:1,000; cat. no. ab692), anti-p53 antibody (1:1,000; cat. no. ab26), anti-Beclin1 antibody (1:1,000; cat. no. ab62557), anti-LC3B antibody (1:1,000; cat. no. ab48394), anti-TWEAK antibody (1:1,000; cat. no. ab37170), anti-p38 (phospho T180+Y182) antibody (1:1,000; cat. no. ab45381), anti-p38 antibody (1:1,000; cat. no. ab31828), and anti-GAPDH antibody (1:2,000; cat. no. ab8245; all Abcam).

    Techniques: Expressing, Western Blot, Activation Assay, Standard Deviation