p38 Search Results


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  • 99
    Millipore p38 mapk inhibitor sb203580
    Effects of JNK or <t>p38</t> <t>MAPK</t> inhibitor on CRP-induced JNK phosphorylation, p38 MAPK phosphorylation, NF-κB p65 phosphorylation and RAGE expression in HCAECs. (A) Western blot analysis of JNK phosphorylation and p38 MAPK phosphorylation in HCAECs treated with vehicle, CRP, JNK inhibitor (SP600125), p38 MAPK inhibitor (SB203580), JNK inhibitor with CRP or p38 MAPK with CRP. (B) RAGE expression in HCAECs. Data are represented as the mean±SEM ( n =3–6). b P
    P38 Mapk Inhibitor Sb203580, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 687 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc p38
    <t>p38</t> MAPK inactivation attenuates scratch-induced astrogliosis in primary astrocyte cultures
    P38, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 14669 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc p38 mapk
    Effects of tylotoin on <t>MAPK</t> signaling pathways. ( A–D ) Western blot showed effects of cathelicidin-NV on ERK, JNK and <t>p38</t> protein kinase phosphorylation ( A ) and relative activation analysis ( B – D ). The results were quantified by ImageJ. The densitometry of phosphorylated ERK, JNK and p38 were normalized to total ERK, JNK and p38, and graphed as the mean ± SEM ( n = 5). The values with treatment by cathelicidin-NV are significantly different from the values for the control (** P
    P38 Mapk, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 7731 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc phospho p38 mapk
    Effects of tylotoin on <t>MAPK</t> signaling pathways. ( A–D ) Western blot showed effects of cathelicidin-NV on ERK, JNK and <t>p38</t> protein kinase phosphorylation ( A ) and relative activation analysis ( B – D ). The results were quantified by ImageJ. The densitometry of phosphorylated ERK, JNK and p38 were normalized to total ERK, JNK and p38, and graphed as the mean ± SEM ( n = 5). The values with treatment by cathelicidin-NV are significantly different from the values for the control (** P
    Phospho P38 Mapk, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 3707 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc anti p p38
    Effect of SFN on the expression of MAPK in the spinal cord from animals with neuropathic pain. Effects of repetitive treatment with 10 mg/kg SFN or vehicle from days 14 to 28 after sciatic nerve injury (CCI) on the protein levels of p-JNK (A) , p-ERK ½ (B) , and <t>p-P38</t> (C) in the spinal cord are represented. The protein levels from Sham-operated (SHAM) mice treated with vehicle has been also represented as controls. In all panels, ∗ indicates significant differences vs. Sham-operated vehicle treated mice and # indicates significant differences vs. CCI-SFN treated mice ( P
    Anti P P38, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1551 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Santa Cruz Biotechnology p38
    Disruption of myeloma cell <t>p38</t> activity has no effect on tumor growth or survival or on the ability of myeloma cells to home to bones. Shown are ( A ) viability of vector-or p38 shRNA-ARP-1 or -MM.1S cells (p38shRNAs) examined byMTT assayat 0 and 24 hour culture; and ( B ) percentages of apoptotic vector- or p38 shRNA-ARP-1 or -MM.1S cells examined by Annexin-V binding assay at 0 and 48 hour cultures. Results represent average values from five independent experiments. ( C ) In situ TUNEL assay results show the percentages of apoptotic CD138 + tumor cells in the bone marrow of SCID mice injected with vector- (vector) or p38 shRNA-ARP-1 or -MM.1S cells. Flow cytometry results show tissue-infiltrating CD138 + human myeloma cells in ( D ) different organs, including bone marrow (BM), spleen, liver, kidney, heart and lung, of SCID mice injected with vector- (vector) or p38 shRNA-ARP-1 or -MM.1S cells or ( E ) bone marrow of SCID mice injected with parental ARP-1 or MM.1S cells and treated with p38 inhibitor SD-169 or PBS. In the experiments, myeloma cells were injected intravenously into SCID mice (10 per each cell line). When circulating M-protein levels reached 10 μg/ml, mice were fed daily with SD-169 (10 mg/kg) or an equal volume of PBS (vehicle controls) for a total of 30 days. After treatment, bone marrow cells were flushed out and CD138 + human myeloma cells were identified with flow cytometry. The results represent average values from three independent experiments of five mice per group.
    P38, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 3208 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/p38/product/Santa Cruz Biotechnology
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    99
    Cell Signaling Technology Inc anti p38 mapk
    Disruption of myeloma cell <t>p38</t> activity has no effect on tumor growth or survival or on the ability of myeloma cells to home to bones. Shown are ( A ) viability of vector-or p38 shRNA-ARP-1 or -MM.1S cells (p38shRNAs) examined byMTT assayat 0 and 24 hour culture; and ( B ) percentages of apoptotic vector- or p38 shRNA-ARP-1 or -MM.1S cells examined by Annexin-V binding assay at 0 and 48 hour cultures. Results represent average values from five independent experiments. ( C ) In situ TUNEL assay results show the percentages of apoptotic CD138 + tumor cells in the bone marrow of SCID mice injected with vector- (vector) or p38 shRNA-ARP-1 or -MM.1S cells. Flow cytometry results show tissue-infiltrating CD138 + human myeloma cells in ( D ) different organs, including bone marrow (BM), spleen, liver, kidney, heart and lung, of SCID mice injected with vector- (vector) or p38 shRNA-ARP-1 or -MM.1S cells or ( E ) bone marrow of SCID mice injected with parental ARP-1 or MM.1S cells and treated with p38 inhibitor SD-169 or PBS. In the experiments, myeloma cells were injected intravenously into SCID mice (10 per each cell line). When circulating M-protein levels reached 10 μg/ml, mice were fed daily with SD-169 (10 mg/kg) or an equal volume of PBS (vehicle controls) for a total of 30 days. After treatment, bone marrow cells were flushed out and CD138 + human myeloma cells were identified with flow cytometry. The results represent average values from three independent experiments of five mice per group.
    Anti P38 Mapk, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1706 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology p38 mapk
    Targeted decreases in <t>p38</t> <t>MAPK</t> expression in neuronal cells attenuate neuronal cell death in rat hippocampal slice cultures. Rat hippocampal slice cultures were transduced with either the AAV-SYN-1-p38 MAPK-AS or the AAV-SYN-1null constructs or were untransduced.
    P38 Mapk, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 1072 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc phosphorylated p38
    Targeted decreases in <t>p38</t> <t>MAPK</t> expression in neuronal cells attenuate neuronal cell death in rat hippocampal slice cultures. Rat hippocampal slice cultures were transduced with either the AAV-SYN-1-p38 MAPK-AS or the AAV-SYN-1null constructs or were untransduced.
    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
    Targeted decreases in <t>p38</t> <t>MAPK</t> expression in neuronal cells attenuate neuronal cell death in rat hippocampal slice cultures. Rat hippocampal slice cultures were transduced with either the AAV-SYN-1-p38 MAPK-AS or the AAV-SYN-1null constructs or were untransduced.
    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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    93
    Santa Cruz Biotechnology anti p38
    Monostability in the <t>p38</t> signaling system. Time course of p38 activation in oocytes treated with 300 mM sorbitol. After 4 h of treatment (arrow), oocytes were washed several times with MBS and maintained in MBS without sorbitol. Pools of 20 oocytes were collected at different times to determine pp38, p38, cytochrome c (CC), and cleaved caspase-3 by Western blot. p38 activity is represented as pp38/p38 ratio, given 100% value to the highest activity. Results are represented as mean ± SEM of three independent experiments. A representative Western blot is shown. Caspase-3 activity was determined as reported before. Data are represented as mean ± SEM (n = 3), *p
    Anti P38, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 1202 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    p38  (Abcam)
    99
    Abcam p38
    Monostability in the <t>p38</t> signaling system. Time course of p38 activation in oocytes treated with 300 mM sorbitol. After 4 h of treatment (arrow), oocytes were washed several times with MBS and maintained in MBS without sorbitol. Pools of 20 oocytes were collected at different times to determine pp38, p38, cytochrome c (CC), and cleaved caspase-3 by Western blot. p38 activity is represented as pp38/p38 ratio, given 100% value to the highest activity. Results are represented as mean ± SEM of three independent experiments. A representative Western blot is shown. Caspase-3 activity was determined as reported before. Data are represented as mean ± SEM (n = 3), *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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    99
    Cell Signaling Technology Inc phosphorylated p38 mapk
    Monostability in the <t>p38</t> signaling system. Time course of p38 activation in oocytes treated with 300 mM sorbitol. After 4 h of treatment (arrow), oocytes were washed several times with MBS and maintained in MBS without sorbitol. Pools of 20 oocytes were collected at different times to determine pp38, p38, cytochrome c (CC), and cleaved caspase-3 by Western blot. p38 activity is represented as pp38/p38 ratio, given 100% value to the highest activity. Results are represented as mean ± SEM of three independent experiments. A representative Western blot is shown. Caspase-3 activity was determined as reported before. Data are represented as mean ± SEM (n = 3), *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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    Image Search Results


    Effects of JNK or p38 MAPK inhibitor on CRP-induced JNK phosphorylation, p38 MAPK phosphorylation, NF-κB p65 phosphorylation and RAGE expression in HCAECs. (A) Western blot analysis of JNK phosphorylation and p38 MAPK phosphorylation in HCAECs treated with vehicle, CRP, JNK inhibitor (SP600125), p38 MAPK inhibitor (SB203580), JNK inhibitor with CRP or p38 MAPK with CRP. (B) RAGE expression in HCAECs. Data are represented as the mean±SEM ( n =3–6). b P

    Journal: Acta Pharmacologica Sinica

    Article Title: C-reactive protein stimulates RAGE expression in human coronary artery endothelial cells in vitro via ROS generation and ERK/NF-κB activation

    doi: 10.1038/aps.2014.163

    Figure Lengend Snippet: Effects of JNK or p38 MAPK inhibitor on CRP-induced JNK phosphorylation, p38 MAPK phosphorylation, NF-κB p65 phosphorylation and RAGE expression in HCAECs. (A) Western blot analysis of JNK phosphorylation and p38 MAPK phosphorylation in HCAECs treated with vehicle, CRP, JNK inhibitor (SP600125), p38 MAPK inhibitor (SB203580), JNK inhibitor with CRP or p38 MAPK with CRP. (B) RAGE expression in HCAECs. Data are represented as the mean±SEM ( n =3–6). b P

    Article Snippet: NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC), ERK inhibitor (PD98059), p38 MAPK inhibitor (SB203580) and JNK inhibitor (SP600125) were purchased from Calbiochem (San Diego, CA, USA).

    Techniques: Expressing, Western Blot

    Role of Fn14 and intracellular signaling pathways in rTWEAK-induced pro-inflammatory cytokine expression. IL-6, IL-8, and MCP-1 protein levels in GO orbital fibroblasts (n = 3) pretreated with ERK kinase (PD98059, 20 μM), JNK (SP600125, 20 μM), p38 MAPK (SB203580, 20 μM), PI3K (LY294002, 20 μM), NF-κB p65 (SC514, 10 μM), and Fn14 (ITEM4, 2.5 μg/ml) inhibitor for 1 h followed by rTWEAK treatment (100 ng/ml, 24 h) were measured by ELISA. Data in columns represent mean ± standard deviation of three experiments. *P

    Journal: PLoS ONE

    Article Title: Tumor necrosis factor-like weak inducer of apoptosis induces inflammation in Graves’ orbital fibroblasts

    doi: 10.1371/journal.pone.0209583

    Figure Lengend Snippet: Role of Fn14 and intracellular signaling pathways in rTWEAK-induced pro-inflammatory cytokine expression. IL-6, IL-8, and MCP-1 protein levels in GO orbital fibroblasts (n = 3) pretreated with ERK kinase (PD98059, 20 μM), JNK (SP600125, 20 μM), p38 MAPK (SB203580, 20 μM), PI3K (LY294002, 20 μM), NF-κB p65 (SC514, 10 μM), and Fn14 (ITEM4, 2.5 μg/ml) inhibitor for 1 h followed by rTWEAK treatment (100 ng/ml, 24 h) were measured by ELISA. Data in columns represent mean ± standard deviation of three experiments. *P

    Article Snippet: Inhibitors of MAPK kinase 1 (PD98059), p38 MAPK (SB203580), JNK (SP600125), phosphoinositide 3-kinase (PI3K; LY294002), and NF-κB p65 (SC514) were purchased from Calbiochem (La Jolla, CA, USA).

    Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Standard Deviation

    Effect of Fn14 inhibitor on rTWEAK-induced hyaluronan production in GO cells. Hyaluronan (ng/ml) level in GO orbital fibroblasts (n = 3) was measured by ELISA after treatment with rTWEAK (10, 100 and 200 ng/ml) for 6 and 24 h. Cells (n = 3) were pretreated with ERK kinase (PD98059, 20 μM), JNK (SP600125, 20 μM), p38 MAPK (SB203580, 20 μM), NF-κB p65 (SC514, 10 μM), and Fn14 (ITEM4, 2.5 μg/ml) inhibitor for 1 h followed by rTWEAK treatment (100 ng/ml, 24 h). Data in columns represent mean ± standard deviation of three experiments. *P

    Journal: PLoS ONE

    Article Title: Tumor necrosis factor-like weak inducer of apoptosis induces inflammation in Graves’ orbital fibroblasts

    doi: 10.1371/journal.pone.0209583

    Figure Lengend Snippet: Effect of Fn14 inhibitor on rTWEAK-induced hyaluronan production in GO cells. Hyaluronan (ng/ml) level in GO orbital fibroblasts (n = 3) was measured by ELISA after treatment with rTWEAK (10, 100 and 200 ng/ml) for 6 and 24 h. Cells (n = 3) were pretreated with ERK kinase (PD98059, 20 μM), JNK (SP600125, 20 μM), p38 MAPK (SB203580, 20 μM), NF-κB p65 (SC514, 10 μM), and Fn14 (ITEM4, 2.5 μg/ml) inhibitor for 1 h followed by rTWEAK treatment (100 ng/ml, 24 h). Data in columns represent mean ± standard deviation of three experiments. *P

    Article Snippet: Inhibitors of MAPK kinase 1 (PD98059), p38 MAPK (SB203580), JNK (SP600125), phosphoinositide 3-kinase (PI3K; LY294002), and NF-κB p65 (SC514) were purchased from Calbiochem (La Jolla, CA, USA).

    Techniques: Enzyme-linked Immunosorbent Assay, 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

    Effects of tylotoin on MAPK signaling pathways. ( A–D ) Western blot showed effects of cathelicidin-NV on ERK, JNK and p38 protein kinase phosphorylation ( A ) and relative activation analysis ( B – D ). The results were quantified by ImageJ. The densitometry of phosphorylated ERK, JNK and p38 were normalized to total ERK, JNK and p38, and graphed as the mean ± SEM ( n = 5). The values with treatment by cathelicidin-NV are significantly different from the values for the control (** P

    Journal: Biochemical Journal

    Article Title: A frog cathelicidin peptide effectively promotes cutaneous wound healing in mice

    doi: 10.1042/BCJ20180286

    Figure Lengend Snippet: Effects of tylotoin on MAPK signaling pathways. ( A–D ) Western blot showed effects of cathelicidin-NV on ERK, JNK and p38 protein kinase phosphorylation ( A ) and relative activation analysis ( B – D ). The results were quantified by ImageJ. The densitometry of phosphorylated ERK, JNK and p38 were normalized to total ERK, JNK and p38, and graphed as the mean ± SEM ( n = 5). The values with treatment by cathelicidin-NV are significantly different from the values for the control (** P

    Article Snippet: Primary antibodies against β-actin (1:5000, Santa Cruz Biotechnology, U.S.A.) and Erk1/2, SAPK/JNK and p38 MAPK (1:2000; Cell Signaling Technology, Beverly, MA, U.S.A.) were used in western blot analysis.

    Techniques: Western Blot, Activation Assay

    Effect of SFN on the expression of MAPK in the spinal cord from animals with neuropathic pain. Effects of repetitive treatment with 10 mg/kg SFN or vehicle from days 14 to 28 after sciatic nerve injury (CCI) on the protein levels of p-JNK (A) , p-ERK ½ (B) , and p-P38 (C) in the spinal cord are represented. The protein levels from Sham-operated (SHAM) mice treated with vehicle has been also represented as controls. In all panels, ∗ indicates significant differences vs. Sham-operated vehicle treated mice and # indicates significant differences vs. CCI-SFN treated mice ( P

    Journal: Frontiers in Pharmacology

    Article Title: Sulforaphane Inhibited the Nociceptive Responses, Anxiety- and Depressive-Like Behaviors Associated With Neuropathic Pain and Improved the Anti-allodynic Effects of Morphine in Mice

    doi: 10.3389/fphar.2018.01332

    Figure Lengend Snippet: Effect of SFN on the expression of MAPK in the spinal cord from animals with neuropathic pain. Effects of repetitive treatment with 10 mg/kg SFN or vehicle from days 14 to 28 after sciatic nerve injury (CCI) on the protein levels of p-JNK (A) , p-ERK ½ (B) , and p-P38 (C) in the spinal cord are represented. The protein levels from Sham-operated (SHAM) mice treated with vehicle has been also represented as controls. In all panels, ∗ indicates significant differences vs. Sham-operated vehicle treated mice and # indicates significant differences vs. CCI-SFN treated mice ( P

    Article Snippet: Proteins were electrophoretically transferred onto PVDF membrane for 120 min, blocked with PBS or TBST + 5% non-fat dry milk or BSA and successively incubated overnight at 4°C with rabbit anti Nrf2 (1:160, Abcam, Cambridge, United Kingdom), HO-1 (1:300, Abcam, Cambridge, United Kingdom), NQO1 (1:333, Sigma, St. Louis, MO, United States), CD11b/c (1:200, Novus Biologicals, Littleton, CO, United States), phospho JNK, total JNK, phospho ERK1/2, total ERK1/2, phospho P38 and total P38 (1:250, Cell Signaling Technology, Danvers, MA, United States), MOR (1:333, Merck, Billerica, MA, United States) or GAPDH antibody (1:5000, Merck, Billerica, MA, United States) which was used as a loading control.

    Techniques: Expressing, Mouse Assay

    Disruption of myeloma cell p38 activity has no effect on tumor growth or survival or on the ability of myeloma cells to home to bones. Shown are ( A ) viability of vector-or p38 shRNA-ARP-1 or -MM.1S cells (p38shRNAs) examined byMTT assayat 0 and 24 hour culture; and ( B ) percentages of apoptotic vector- or p38 shRNA-ARP-1 or -MM.1S cells examined by Annexin-V binding assay at 0 and 48 hour cultures. Results represent average values from five independent experiments. ( C ) In situ TUNEL assay results show the percentages of apoptotic CD138 + tumor cells in the bone marrow of SCID mice injected with vector- (vector) or p38 shRNA-ARP-1 or -MM.1S cells. Flow cytometry results show tissue-infiltrating CD138 + human myeloma cells in ( D ) different organs, including bone marrow (BM), spleen, liver, kidney, heart and lung, of SCID mice injected with vector- (vector) or p38 shRNA-ARP-1 or -MM.1S cells or ( E ) bone marrow of SCID mice injected with parental ARP-1 or MM.1S cells and treated with p38 inhibitor SD-169 or PBS. In the experiments, myeloma cells were injected intravenously into SCID mice (10 per each cell line). When circulating M-protein levels reached 10 μg/ml, mice were fed daily with SD-169 (10 mg/kg) or an equal volume of PBS (vehicle controls) for a total of 30 days. After treatment, bone marrow cells were flushed out and CD138 + human myeloma cells were identified with flow cytometry. The results represent average values from three independent experiments of five mice per group.

    Journal: Leukemia : official journal of the Leukemia Society of America, Leukemia Research Fund, U.K

    Article Title: Constitutive activation of p38 MAPK in tumor cells contributes to osteolytic bone lesions in multiple myeloma

    doi: 10.1038/leu.2012.71

    Figure Lengend Snippet: Disruption of myeloma cell p38 activity has no effect on tumor growth or survival or on the ability of myeloma cells to home to bones. Shown are ( A ) viability of vector-or p38 shRNA-ARP-1 or -MM.1S cells (p38shRNAs) examined byMTT assayat 0 and 24 hour culture; and ( B ) percentages of apoptotic vector- or p38 shRNA-ARP-1 or -MM.1S cells examined by Annexin-V binding assay at 0 and 48 hour cultures. Results represent average values from five independent experiments. ( C ) In situ TUNEL assay results show the percentages of apoptotic CD138 + tumor cells in the bone marrow of SCID mice injected with vector- (vector) or p38 shRNA-ARP-1 or -MM.1S cells. Flow cytometry results show tissue-infiltrating CD138 + human myeloma cells in ( D ) different organs, including bone marrow (BM), spleen, liver, kidney, heart and lung, of SCID mice injected with vector- (vector) or p38 shRNA-ARP-1 or -MM.1S cells or ( E ) bone marrow of SCID mice injected with parental ARP-1 or MM.1S cells and treated with p38 inhibitor SD-169 or PBS. In the experiments, myeloma cells were injected intravenously into SCID mice (10 per each cell line). When circulating M-protein levels reached 10 μg/ml, mice were fed daily with SD-169 (10 mg/kg) or an equal volume of PBS (vehicle controls) for a total of 30 days. After treatment, bone marrow cells were flushed out and CD138 + human myeloma cells were identified with flow cytometry. The results represent average values from three independent experiments of five mice per group.

    Article Snippet: Plamids and reagents shRNAs for p38 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and packed into retroviral vector pSIREN-RetroQ (BD Biosciences Clontech, Mountain View, CA).

    Techniques: Activity Assay, Plasmid Preparation, shRNA, Binding Assay, In Situ, TUNEL Assay, Mouse Assay, Injection, Flow Cytometry, Cytometry

    Activated tumor cell p38 enhances osteoclastogenesis in vivo . Shown are ( A ) representative images and numbers of TRAP-positive osteoclasts on murine trabecular bone (scale bars in larger windows: 50 μm; in smaller windows: 10 μm), and quantitative analysis of osteoclast parameters such as ( B ) percentages of osteoclast-mediated erosion of bone surface (ES/BS) and ( C ) numbers of osteoclasts present on bone surface (Oc. S/BS) in bone sections of SCID mice bearing vector- or p38 shRNA-ARP-1 (p38shRNAs/p38SH) myeloma at week 8 after tumor inoculation. Shown are levels of circulating ( D ) CTX-1, ( E ) TRAP5b, and ( F ) RANKL in mice injected with vector- or p38 shRNA-ARP-1 cells at 0 week (0w) or 8 weeks (8w) after tumor inoculation. ( G ) Immunohistochemical staining shows the numbers of TRAP-positive osteoclasts present on human trabecular bone surfaces of primary myeloma-bearing SCID-hu mice treated without (vehicle) or with p38 inhibitor SD-169 at week 8 after tumor injection. Arrows indicate osteoclasts. Representative results from four independent experiments are shown. ** P ≤ 0.01; *** P ≤ 0.001.

    Journal: Leukemia : official journal of the Leukemia Society of America, Leukemia Research Fund, U.K

    Article Title: Constitutive activation of p38 MAPK in tumor cells contributes to osteolytic bone lesions in multiple myeloma

    doi: 10.1038/leu.2012.71

    Figure Lengend Snippet: Activated tumor cell p38 enhances osteoclastogenesis in vivo . Shown are ( A ) representative images and numbers of TRAP-positive osteoclasts on murine trabecular bone (scale bars in larger windows: 50 μm; in smaller windows: 10 μm), and quantitative analysis of osteoclast parameters such as ( B ) percentages of osteoclast-mediated erosion of bone surface (ES/BS) and ( C ) numbers of osteoclasts present on bone surface (Oc. S/BS) in bone sections of SCID mice bearing vector- or p38 shRNA-ARP-1 (p38shRNAs/p38SH) myeloma at week 8 after tumor inoculation. Shown are levels of circulating ( D ) CTX-1, ( E ) TRAP5b, and ( F ) RANKL in mice injected with vector- or p38 shRNA-ARP-1 cells at 0 week (0w) or 8 weeks (8w) after tumor inoculation. ( G ) Immunohistochemical staining shows the numbers of TRAP-positive osteoclasts present on human trabecular bone surfaces of primary myeloma-bearing SCID-hu mice treated without (vehicle) or with p38 inhibitor SD-169 at week 8 after tumor injection. Arrows indicate osteoclasts. Representative results from four independent experiments are shown. ** P ≤ 0.01; *** P ≤ 0.001.

    Article Snippet: Plamids and reagents shRNAs for p38 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and packed into retroviral vector pSIREN-RetroQ (BD Biosciences Clontech, Mountain View, CA).

    Techniques: In Vivo, Mouse Assay, Plasmid Preparation, shRNA, Injection, Immunohistochemistry, Staining

    Myeloma cells with high/detectable p38 activity cause bone lesions in mouse models. ( A ) Representative radiographic images show lytic bone lesions in the implanted human bones of SCID-hu mice bearing primary myeloma cells from one (patient 10; pt10) of four patients with low or undetectable p38 or one (patient 9; pt9) of eight patients with high/detectable p38 activity. ( B ) Percentages of mice with bone lesions, detected by radiographs, at 4 and 8 weeks after injection ofprimary myeloma cells with high/detectable p38 activity from eight patients or with undetectable pp38 activity from four patients. ( C ) Western blot analysis shows the levels of phosphorylated p38 (pp38) and nonphosphorylated p38 (p38) and AF-2 in MM1-144, MM.1S, and ARP-1 myeloma cells. ( D ) Representative radiographic images show lytic bone lesions in the distal femurs of SCID mice (10 per each cell line) injected with ARP-1, MM.1S, or MM1-144 myeloma cells. Tumor-free mice (no tumor) served as controls. Red arrows indicate osteolytic lesions. ( E ) Representative μ-CT images (upper panels) and histologic examinations (lower panels) and ( F ) quantitative analyses show the trabecular bone volume density (BV/TV) in distal femurs of SCID mice bearing ARP-1, MM.1S, or MM1-144 myeloma. ** P

    Journal: Leukemia : official journal of the Leukemia Society of America, Leukemia Research Fund, U.K

    Article Title: Constitutive activation of p38 MAPK in tumor cells contributes to osteolytic bone lesions in multiple myeloma

    doi: 10.1038/leu.2012.71

    Figure Lengend Snippet: Myeloma cells with high/detectable p38 activity cause bone lesions in mouse models. ( A ) Representative radiographic images show lytic bone lesions in the implanted human bones of SCID-hu mice bearing primary myeloma cells from one (patient 10; pt10) of four patients with low or undetectable p38 or one (patient 9; pt9) of eight patients with high/detectable p38 activity. ( B ) Percentages of mice with bone lesions, detected by radiographs, at 4 and 8 weeks after injection ofprimary myeloma cells with high/detectable p38 activity from eight patients or with undetectable pp38 activity from four patients. ( C ) Western blot analysis shows the levels of phosphorylated p38 (pp38) and nonphosphorylated p38 (p38) and AF-2 in MM1-144, MM.1S, and ARP-1 myeloma cells. ( D ) Representative radiographic images show lytic bone lesions in the distal femurs of SCID mice (10 per each cell line) injected with ARP-1, MM.1S, or MM1-144 myeloma cells. Tumor-free mice (no tumor) served as controls. Red arrows indicate osteolytic lesions. ( E ) Representative μ-CT images (upper panels) and histologic examinations (lower panels) and ( F ) quantitative analyses show the trabecular bone volume density (BV/TV) in distal femurs of SCID mice bearing ARP-1, MM.1S, or MM1-144 myeloma. ** P

    Article Snippet: Plamids and reagents shRNAs for p38 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and packed into retroviral vector pSIREN-RetroQ (BD Biosciences Clontech, Mountain View, CA).

    Techniques: Activity Assay, Mouse Assay, Injection, Western Blot

    Administration of p38 inhibitor reduces myeloma-induced osteolytic bone lesions. In vivo injection of p38 specific inhibitor SD-169 significantly reduced myeloma-induced bone lesions in fetal human bones implanted into SCID-hu mice bearing a primary myeloma xenograft; lesions were detected by ( A ) radiography and ( B ) μ-CT scanning. ( C ) SD-169 also reduced bone lesions in distal femurs of ARP-1- or MM.1S-bearing SCID mice. ( D ) Tumor burden was measured as circulating human Ig in SCID mice inoculated with ARP-1 or MM.1S cells treated without or with p38 inhibitor SD-169. CD138 + primary myeloma cells were isolated from three myeloma patients (Pt1, Pt2, and Pt3) with high/detectable p38 activity and injected into the implanted human bones of SCID-hu mice. Myeloma ARP-1 or MM.1S cells were injected intravenously into SCID mice (10 per each cell line). When circulating M-protein levels reached 10 μg/ml, mice were fed daily with SD-169 (10 mg/kg) or an equal volume of PBS (vehicle controls) for a total of 30 days. Arrows indicate osteolytic bone lesions.

    Journal: Leukemia : official journal of the Leukemia Society of America, Leukemia Research Fund, U.K

    Article Title: Constitutive activation of p38 MAPK in tumor cells contributes to osteolytic bone lesions in multiple myeloma

    doi: 10.1038/leu.2012.71

    Figure Lengend Snippet: Administration of p38 inhibitor reduces myeloma-induced osteolytic bone lesions. In vivo injection of p38 specific inhibitor SD-169 significantly reduced myeloma-induced bone lesions in fetal human bones implanted into SCID-hu mice bearing a primary myeloma xenograft; lesions were detected by ( A ) radiography and ( B ) μ-CT scanning. ( C ) SD-169 also reduced bone lesions in distal femurs of ARP-1- or MM.1S-bearing SCID mice. ( D ) Tumor burden was measured as circulating human Ig in SCID mice inoculated with ARP-1 or MM.1S cells treated without or with p38 inhibitor SD-169. CD138 + primary myeloma cells were isolated from three myeloma patients (Pt1, Pt2, and Pt3) with high/detectable p38 activity and injected into the implanted human bones of SCID-hu mice. Myeloma ARP-1 or MM.1S cells were injected intravenously into SCID mice (10 per each cell line). When circulating M-protein levels reached 10 μg/ml, mice were fed daily with SD-169 (10 mg/kg) or an equal volume of PBS (vehicle controls) for a total of 30 days. Arrows indicate osteolytic bone lesions.

    Article Snippet: Plamids and reagents shRNAs for p38 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and packed into retroviral vector pSIREN-RetroQ (BD Biosciences Clontech, Mountain View, CA).

    Techniques: In Vivo, Injection, Mouse Assay, Isolation, Activity Assay

    Knockdown of tumor cell p38 activity by shRNAs reduces myeloma-induced osteolytic bone lesions. Myeloma cells were stably transfected with retrovirus containing control vector or p38-shRNAs to knock down p38 activity. Western blot analysis shows ( A ) levels of phosphorylated p38 (pp38), nonphosphorylated p38 (p38), nonphosphorylated or phosphorylated AF-2 (a p38 downstream kinase), and nonphosphorylated or phosphorylated ERK1/2 in vector-(vector) or p38 shRNA-ARP-1 or -MM.1S cells, and ( B ) levels of phosphorylated MKK3/6 (pMKK3/6, upstream kinase of p38), pp38, and phosphorylated MAPKPK-2 (pMK-2, downstream kinase of p38) in vector- or p38 shRNA-ARP-1 (p38shRNAs) or -MM.1S (data not shown) cells treated with 10 nM of anisomycin (Aniso) at different time points (0, 15, 30, 60 minutes). β-actin protein levels served as loading controls. ( C ) Immunohistochemical staining shows the expression of p38, pp38, and CD138 (myeloma cell marker) in bone sections of SCID mice injected with vector- (vector) or p38 shRNA-ARP-1 cells. The vector-ARP-1 or p38 shRNA-ARP-1 cells were intravenously injected into SCID mice. Sections of distal femur taken from mice killed 4 weeks after tumor inoculation were immunostained with antibodies against p38, pp38, and CD138. Scale bar, 10 μM. ( D ) Representative radiographic images (upper panels) show osteolytic lesions in the distal femurs and histologic examinations (lower panels) show the densities of trabecular bones in the bone marrow of SCID mice (10 per group) injected with vector- or p38 shRNA-ARP-1 cells and killed 8 weeks after tumor injection. Arrows indicate osteolytic bone lesions. ( E ) Percentages of mice with bone lesions, detected by radiographs, are shown at different time intervals after injection with vector- or p38shRNA-ARP-1 cells. Analysis of bone sections by μ-CT scanning shows ( F ) greater trabecular bone volumes and ( G ) larger densities inthe distal femurs of SCID mice injected with p38 shRNA-ARP-1 cells than in those injected with vector-ARP-1 cells. ** P ≤ 0.01; *** P ≤ 0.001.

    Journal: Leukemia : official journal of the Leukemia Society of America, Leukemia Research Fund, U.K

    Article Title: Constitutive activation of p38 MAPK in tumor cells contributes to osteolytic bone lesions in multiple myeloma

    doi: 10.1038/leu.2012.71

    Figure Lengend Snippet: Knockdown of tumor cell p38 activity by shRNAs reduces myeloma-induced osteolytic bone lesions. Myeloma cells were stably transfected with retrovirus containing control vector or p38-shRNAs to knock down p38 activity. Western blot analysis shows ( A ) levels of phosphorylated p38 (pp38), nonphosphorylated p38 (p38), nonphosphorylated or phosphorylated AF-2 (a p38 downstream kinase), and nonphosphorylated or phosphorylated ERK1/2 in vector-(vector) or p38 shRNA-ARP-1 or -MM.1S cells, and ( B ) levels of phosphorylated MKK3/6 (pMKK3/6, upstream kinase of p38), pp38, and phosphorylated MAPKPK-2 (pMK-2, downstream kinase of p38) in vector- or p38 shRNA-ARP-1 (p38shRNAs) or -MM.1S (data not shown) cells treated with 10 nM of anisomycin (Aniso) at different time points (0, 15, 30, 60 minutes). β-actin protein levels served as loading controls. ( C ) Immunohistochemical staining shows the expression of p38, pp38, and CD138 (myeloma cell marker) in bone sections of SCID mice injected with vector- (vector) or p38 shRNA-ARP-1 cells. The vector-ARP-1 or p38 shRNA-ARP-1 cells were intravenously injected into SCID mice. Sections of distal femur taken from mice killed 4 weeks after tumor inoculation were immunostained with antibodies against p38, pp38, and CD138. Scale bar, 10 μM. ( D ) Representative radiographic images (upper panels) show osteolytic lesions in the distal femurs and histologic examinations (lower panels) show the densities of trabecular bones in the bone marrow of SCID mice (10 per group) injected with vector- or p38 shRNA-ARP-1 cells and killed 8 weeks after tumor injection. Arrows indicate osteolytic bone lesions. ( E ) Percentages of mice with bone lesions, detected by radiographs, are shown at different time intervals after injection with vector- or p38shRNA-ARP-1 cells. Analysis of bone sections by μ-CT scanning shows ( F ) greater trabecular bone volumes and ( G ) larger densities inthe distal femurs of SCID mice injected with p38 shRNA-ARP-1 cells than in those injected with vector-ARP-1 cells. ** P ≤ 0.01; *** P ≤ 0.001.

    Article Snippet: Plamids and reagents shRNAs for p38 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and packed into retroviral vector pSIREN-RetroQ (BD Biosciences Clontech, Mountain View, CA).

    Techniques: Activity Assay, Stable Transfection, Transfection, Plasmid Preparation, Western Blot, shRNA, Immunohistochemistry, Staining, Expressing, Marker, Mouse Assay, Injection

    Activated tumor cell p38 inhibits osteoblastogenesis in vivo . Shown are ( A ) representative images of toluidine blue staining showing the numbers of osteoblasts, and the results of quantitative analysis of osteoblast parameters, including ( B ) total osteoblast numbers on the surface and ( C ) osteoid percentage of trabecular bone of SCID mice bearing vector- or p38 shRNA-ARP-1 myeloma. ELISA showed levels of circulating ( D ) osteocalcin or ( E ) ALP in mice prior to (week 0; 0w) and 8 weeks (8w) after injection with controls cells or p38 shRNA-ARP-1 cells. ( F ) Immunohistochemical staining shows the numbers of osteoblasts present at week 8 on the surface of human trabecular bone implanted into primary myeloma-xenografted SCID-hu mice treated without (vehicle) or with p38 inhibitor SD-169. Arrows indicate osteoblasts. Generation of osteoblasts from normal mesenchymal stem cells in osteoblast medium with addition of conditioning medium of vector or p38 shRNA-ARP-1 cells (p38SH). Representative results from four independent experiments are shown. ** P ≤ 0.01; *** P ≤ 0.001.

    Journal: Leukemia : official journal of the Leukemia Society of America, Leukemia Research Fund, U.K

    Article Title: Constitutive activation of p38 MAPK in tumor cells contributes to osteolytic bone lesions in multiple myeloma

    doi: 10.1038/leu.2012.71

    Figure Lengend Snippet: Activated tumor cell p38 inhibits osteoblastogenesis in vivo . Shown are ( A ) representative images of toluidine blue staining showing the numbers of osteoblasts, and the results of quantitative analysis of osteoblast parameters, including ( B ) total osteoblast numbers on the surface and ( C ) osteoid percentage of trabecular bone of SCID mice bearing vector- or p38 shRNA-ARP-1 myeloma. ELISA showed levels of circulating ( D ) osteocalcin or ( E ) ALP in mice prior to (week 0; 0w) and 8 weeks (8w) after injection with controls cells or p38 shRNA-ARP-1 cells. ( F ) Immunohistochemical staining shows the numbers of osteoblasts present at week 8 on the surface of human trabecular bone implanted into primary myeloma-xenografted SCID-hu mice treated without (vehicle) or with p38 inhibitor SD-169. Arrows indicate osteoblasts. Generation of osteoblasts from normal mesenchymal stem cells in osteoblast medium with addition of conditioning medium of vector or p38 shRNA-ARP-1 cells (p38SH). Representative results from four independent experiments are shown. ** P ≤ 0.01; *** P ≤ 0.001.

    Article Snippet: Plamids and reagents shRNAs for p38 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and packed into retroviral vector pSIREN-RetroQ (BD Biosciences Clontech, Mountain View, CA).

    Techniques: In Vivo, Staining, Mouse Assay, Plasmid Preparation, shRNA, Enzyme-linked Immunosorbent Assay, ALP Assay, Injection, Immunohistochemistry

    Constitutive activation of p38 in myeloma cells. Representative images of immunohistochemical staining for pp38 in ( A ) bone marrow biopsy specimens of 10 randomly selected patients with newly diagnosed myeloma (Pt1–Pt10) and ( B ) a tissue array containing bone marrow samples of 10 myeloma patients (Pt1–Pt10) and 11 healthy donors. (Only five samples, N1–N5; are shown. The other six samples also stained negatively for pp38.) Scale bar, 10 μM. Western blot analysis showing the levels of phosphorylated p38 (pp38) and nonphosphorylated p38 (p38) in ( C ) six myeloma cell lines and in ( D ) three normal PBMCs. Representative results of three independent experiments are shown.

    Journal: Leukemia : official journal of the Leukemia Society of America, Leukemia Research Fund, U.K

    Article Title: Constitutive activation of p38 MAPK in tumor cells contributes to osteolytic bone lesions in multiple myeloma

    doi: 10.1038/leu.2012.71

    Figure Lengend Snippet: Constitutive activation of p38 in myeloma cells. Representative images of immunohistochemical staining for pp38 in ( A ) bone marrow biopsy specimens of 10 randomly selected patients with newly diagnosed myeloma (Pt1–Pt10) and ( B ) a tissue array containing bone marrow samples of 10 myeloma patients (Pt1–Pt10) and 11 healthy donors. (Only five samples, N1–N5; are shown. The other six samples also stained negatively for pp38.) Scale bar, 10 μM. Western blot analysis showing the levels of phosphorylated p38 (pp38) and nonphosphorylated p38 (p38) in ( C ) six myeloma cell lines and in ( D ) three normal PBMCs. Representative results of three independent experiments are shown.

    Article Snippet: Plamids and reagents shRNAs for p38 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and packed into retroviral vector pSIREN-RetroQ (BD Biosciences Clontech, Mountain View, CA).

    Techniques: Activation Assay, Immunohistochemistry, Staining, Western Blot

    Activations of candidate proteins involved in P4-induced increases in the levels of p21 cip1 and p27 kip1 protein in RASMCs. Treatment of RASMCs with P4 (50 nM) for 5 min increased the levels of p-cSrc, p-Raf-1, p-AKT, p-ERK1/2, and p-p38 protein (A) and membrane translocation of Kras, an indicator for Kras activation (B). Data are representative of 2 independent experiments with similar results. Values shown in parentheses represent the quantified results adjusted with their own total protein level (A) or with G3PDH (for cytosol protein) and cadherin (for membrane protein), and expressed as ratio over control. Con, control.

    Journal: PLoS ONE

    Article Title: Extra-Nuclear Signaling Pathway Involved in Progesterone-Induced Up-Regulations of p21cip1 and p27kip1 in Male Rat Aortic Smooth Muscle Cells

    doi: 10.1371/journal.pone.0125903

    Figure Lengend Snippet: Activations of candidate proteins involved in P4-induced increases in the levels of p21 cip1 and p27 kip1 protein in RASMCs. Treatment of RASMCs with P4 (50 nM) for 5 min increased the levels of p-cSrc, p-Raf-1, p-AKT, p-ERK1/2, and p-p38 protein (A) and membrane translocation of Kras, an indicator for Kras activation (B). Data are representative of 2 independent experiments with similar results. Values shown in parentheses represent the quantified results adjusted with their own total protein level (A) or with G3PDH (for cytosol protein) and cadherin (for membrane protein), and expressed as ratio over control. Con, control.

    Article Snippet: Reagents Antibodies against p-AKT, AKT, cadherin, p-ERK, p21cip1 , p-p38, p38, p-Raf-1, PARP, p65, p-IκBα, or IκBα were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

    Techniques: Translocation Assay, Activation Assay

    Model for P4-induced up-regulations in the levels of p21 cip1 and p27 kip1 protein in RASMCs. P4 activated cSrc, subsequently causing membrane translocation of Kras, which in turn activated Raf-1/AKT/ERK1/2/p38/IκBα and increased nuclear translocation of NFκB (p65), and eventually increasing the levels of p21 cip1 and p27 kip1 protein through up-regulating p53 expression in RASMCs. PR, progesterone receptor.

    Journal: PLoS ONE

    Article Title: Extra-Nuclear Signaling Pathway Involved in Progesterone-Induced Up-Regulations of p21cip1 and p27kip1 in Male Rat Aortic Smooth Muscle Cells

    doi: 10.1371/journal.pone.0125903

    Figure Lengend Snippet: Model for P4-induced up-regulations in the levels of p21 cip1 and p27 kip1 protein in RASMCs. P4 activated cSrc, subsequently causing membrane translocation of Kras, which in turn activated Raf-1/AKT/ERK1/2/p38/IκBα and increased nuclear translocation of NFκB (p65), and eventually increasing the levels of p21 cip1 and p27 kip1 protein through up-regulating p53 expression in RASMCs. PR, progesterone receptor.

    Article Snippet: Reagents Antibodies against p-AKT, AKT, cadherin, p-ERK, p21cip1 , p-p38, p38, p-Raf-1, PARP, p65, p-IκBα, or IκBα were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

    Techniques: Translocation Assay, Expressing

    The cSrc/Kras/Raf-1/AKT/ERK/p38-mediated pathway is involved in P4-induced NFκB (p65) nuclear translocation. (A) Pre-treatment of RASMCs with PP2 (200 nM) for 1 h prevented the P4-induced NFκB nuclear translocation. Pre-treatment of RASMCs with 200 nM PP2 (B), 1 μM SB 203580 (C), or 100 nM wortmannin (D) prevented P4-induced increases in the levels of p-IκBα. (E) Pre-transfection with dn-AKT cDNA prevented P4-induced increases the levels of p53, p21 cip1 and p27 kip1 protein. (F) Pre-treatment with BAY (10 nM) prevented P4-induced increases in the levels of p53, p21 cip1 and p27 kip1 protein. (A-F) Data are representative of 2 independent experiments with similar results. Values shown in parentheses represent the quantified results adjusted with their own total protein level, RARP (for nuclear protein) or G3PDH (for cytosolic protein) and expressed as ratio over control. (G) Pre-transfection with dn-p53 cDNA prevented P4-induced increases in the levels of p21 cip1 and p27 kip1 protein. Values shown in parentheses represent the quantified results adjusted with α-tubulin protein level and expressed as ratio over control. Values represent the means±s.e.mean. (n = 3). * P

    Journal: PLoS ONE

    Article Title: Extra-Nuclear Signaling Pathway Involved in Progesterone-Induced Up-Regulations of p21cip1 and p27kip1 in Male Rat Aortic Smooth Muscle Cells

    doi: 10.1371/journal.pone.0125903

    Figure Lengend Snippet: The cSrc/Kras/Raf-1/AKT/ERK/p38-mediated pathway is involved in P4-induced NFκB (p65) nuclear translocation. (A) Pre-treatment of RASMCs with PP2 (200 nM) for 1 h prevented the P4-induced NFκB nuclear translocation. Pre-treatment of RASMCs with 200 nM PP2 (B), 1 μM SB 203580 (C), or 100 nM wortmannin (D) prevented P4-induced increases in the levels of p-IκBα. (E) Pre-transfection with dn-AKT cDNA prevented P4-induced increases the levels of p53, p21 cip1 and p27 kip1 protein. (F) Pre-treatment with BAY (10 nM) prevented P4-induced increases in the levels of p53, p21 cip1 and p27 kip1 protein. (A-F) Data are representative of 2 independent experiments with similar results. Values shown in parentheses represent the quantified results adjusted with their own total protein level, RARP (for nuclear protein) or G3PDH (for cytosolic protein) and expressed as ratio over control. (G) Pre-transfection with dn-p53 cDNA prevented P4-induced increases in the levels of p21 cip1 and p27 kip1 protein. Values shown in parentheses represent the quantified results adjusted with α-tubulin protein level and expressed as ratio over control. Values represent the means±s.e.mean. (n = 3). * P

    Article Snippet: Reagents Antibodies against p-AKT, AKT, cadherin, p-ERK, p21cip1 , p-p38, p38, p-Raf-1, PARP, p65, p-IκBα, or IκBα were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

    Techniques: Translocation Assay, Transfection

    cSrc is the most upstream molecule involved in P4-induced increases in the levels of p21 cip1 and p27 kip1 . Pre-treatment with PP2 (200 nM) for 1 h prevented P4-induced membrane translocation of Kras (A), increases in the levels of p-Raf-1, p-ERK1/2, p-AKT and p-p38 protein (B), and p21 cip1 , p27 kip1 and p53 protein (C). Data are representative of 2 independent experiments with similar results. Values shown in parentheses represent the quantified results adjusted with G3PDH and cadherin for cytosol and membrane, respectively (A), with their own total protein level (B), or with α-tubulin (C) and expressed as ratio over control. Con, control.

    Journal: PLoS ONE

    Article Title: Extra-Nuclear Signaling Pathway Involved in Progesterone-Induced Up-Regulations of p21cip1 and p27kip1 in Male Rat Aortic Smooth Muscle Cells

    doi: 10.1371/journal.pone.0125903

    Figure Lengend Snippet: cSrc is the most upstream molecule involved in P4-induced increases in the levels of p21 cip1 and p27 kip1 . Pre-treatment with PP2 (200 nM) for 1 h prevented P4-induced membrane translocation of Kras (A), increases in the levels of p-Raf-1, p-ERK1/2, p-AKT and p-p38 protein (B), and p21 cip1 , p27 kip1 and p53 protein (C). Data are representative of 2 independent experiments with similar results. Values shown in parentheses represent the quantified results adjusted with G3PDH and cadherin for cytosol and membrane, respectively (A), with their own total protein level (B), or with α-tubulin (C) and expressed as ratio over control. Con, control.

    Article Snippet: Reagents Antibodies against p-AKT, AKT, cadherin, p-ERK, p21cip1 , p-p38, p38, p-Raf-1, PARP, p65, p-IκBα, or IκBα were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

    Techniques: Translocation Assay

    Involvement of AKT/ERK/p38 in P4-induced increases in the levels of p21 cip1 and p27 kip1 protein in RASMCs. (A) Pre-transfection of RASMCs with dn-ERK2 cDNA prevented P4-induced increases in the levels of p-ERK1/2, and p-p38 protein, but not p-Raf-1 and p-AKT protein. (B) Pre-transfection of RASMCs with dn-AKT cDNA prevented P4-induced increases in the levels of p-AKT, pERK1/2, and p-p38 protein. (C) Pre-treatment of RASMCs with a p38 inhibitor, SB 203580 (1 μM), for 1 h prevented P4-induced increases in the level of p-p38 protein, but not p-AKT and p-ERK1/2 protein. Data are representative of 2 independent experiments with similar results. Values shown in parentheses represent the quantified results adjusted with their own total protein level and expressed as ratio over control. Con, control; dn, dominate negative; SB, SB 203580.

    Journal: PLoS ONE

    Article Title: Extra-Nuclear Signaling Pathway Involved in Progesterone-Induced Up-Regulations of p21cip1 and p27kip1 in Male Rat Aortic Smooth Muscle Cells

    doi: 10.1371/journal.pone.0125903

    Figure Lengend Snippet: Involvement of AKT/ERK/p38 in P4-induced increases in the levels of p21 cip1 and p27 kip1 protein in RASMCs. (A) Pre-transfection of RASMCs with dn-ERK2 cDNA prevented P4-induced increases in the levels of p-ERK1/2, and p-p38 protein, but not p-Raf-1 and p-AKT protein. (B) Pre-transfection of RASMCs with dn-AKT cDNA prevented P4-induced increases in the levels of p-AKT, pERK1/2, and p-p38 protein. (C) Pre-treatment of RASMCs with a p38 inhibitor, SB 203580 (1 μM), for 1 h prevented P4-induced increases in the level of p-p38 protein, but not p-AKT and p-ERK1/2 protein. Data are representative of 2 independent experiments with similar results. Values shown in parentheses represent the quantified results adjusted with their own total protein level and expressed as ratio over control. Con, control; dn, dominate negative; SB, SB 203580.

    Article Snippet: Reagents Antibodies against p-AKT, AKT, cadherin, p-ERK, p21cip1 , p-p38, p38, p-Raf-1, PARP, p65, p-IκBα, or IκBα were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

    Techniques: Transfection

    Kras and Raf-1 are the upstream molecules of the AKT/ERK1/2/p38 pathway involved in P4-induced increases in the levels of p21 cip1 and p27 kip1 protein in RASMCs. (A) Pre-treatment of RASMCs with a Kras inhibitor, FTI (10 μM), for 1 h prevented P4-induced increases in the levels of p-Raf-1, p-ERK1/2, p-AKT, and p-p38 protein. (B) Pre-treatment of RASMCs with a Raf-1 inhibitor, sulindac sulfide (10 μM), for 1 h prevented P4-induced increases in the levels of p-ERK1/2, p-AKT, and p-p38 protein. Data are representative of 2 independent experiments with similar results. Values shown in parentheses represent the quantified results adjusted with their own total protein level and expressed as ratio over control. Con, control. FTI, farnesyltransferase inhibitor; SS, sulindac sulfide.

    Journal: PLoS ONE

    Article Title: Extra-Nuclear Signaling Pathway Involved in Progesterone-Induced Up-Regulations of p21cip1 and p27kip1 in Male Rat Aortic Smooth Muscle Cells

    doi: 10.1371/journal.pone.0125903

    Figure Lengend Snippet: Kras and Raf-1 are the upstream molecules of the AKT/ERK1/2/p38 pathway involved in P4-induced increases in the levels of p21 cip1 and p27 kip1 protein in RASMCs. (A) Pre-treatment of RASMCs with a Kras inhibitor, FTI (10 μM), for 1 h prevented P4-induced increases in the levels of p-Raf-1, p-ERK1/2, p-AKT, and p-p38 protein. (B) Pre-treatment of RASMCs with a Raf-1 inhibitor, sulindac sulfide (10 μM), for 1 h prevented P4-induced increases in the levels of p-ERK1/2, p-AKT, and p-p38 protein. Data are representative of 2 independent experiments with similar results. Values shown in parentheses represent the quantified results adjusted with their own total protein level and expressed as ratio over control. Con, control. FTI, farnesyltransferase inhibitor; SS, sulindac sulfide.

    Article Snippet: Reagents Antibodies against p-AKT, AKT, cadherin, p-ERK, p21cip1 , p-p38, p38, p-Raf-1, PARP, p65, p-IκBα, or IκBα were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

    Techniques:

    Targeted decreases in p38 MAPK expression in neuronal cells attenuate neuronal cell death in rat hippocampal slice cultures. Rat hippocampal slice cultures were transduced with either the AAV-SYN-1-p38 MAPK-AS or the AAV-SYN-1null constructs or were untransduced.

    Journal: The European journal of neuroscience

    Article Title: Increased p38 mitogen-activated protein kinase signaling is involved in the oxidative stress associated with oxygen and glucose deprivation in neonatal hippocampal slice cultures

    doi: 10.1111/j.1460-9568.2011.07786.x

    Figure Lengend Snippet: Targeted decreases in p38 MAPK expression in neuronal cells attenuate neuronal cell death in rat hippocampal slice cultures. Rat hippocampal slice cultures were transduced with either the AAV-SYN-1-p38 MAPK-AS or the AAV-SYN-1null constructs or were untransduced.

    Article Snippet: The primary antibodies used were anti-phospho-p38 MAPK and p38 MAPK (Santa Cruz, CA, USA), anti-caspase-3 and anti-cleaved caspase-3 (Cell Signaling, Danvers, MA, USA).

    Techniques: Expressing, Transduction, Construct

    OGD increases apoptotic nuclei in the neurons of rat hippocampal slice cultures. Rat hippocampal slice cultures were exposed to OGD in the presence or absence of the p38 MAPK inhibitor, SB203580 (50 μ m , 2 h prior to OGD). Slices were harvested

    Journal: The European journal of neuroscience

    Article Title: Increased p38 mitogen-activated protein kinase signaling is involved in the oxidative stress associated with oxygen and glucose deprivation in neonatal hippocampal slice cultures

    doi: 10.1111/j.1460-9568.2011.07786.x

    Figure Lengend Snippet: OGD increases apoptotic nuclei in the neurons of rat hippocampal slice cultures. Rat hippocampal slice cultures were exposed to OGD in the presence or absence of the p38 MAPK inhibitor, SB203580 (50 μ m , 2 h prior to OGD). Slices were harvested

    Article Snippet: The primary antibodies used were anti-phospho-p38 MAPK and p38 MAPK (Santa Cruz, CA, USA), anti-caspase-3 and anti-cleaved caspase-3 (Cell Signaling, Danvers, MA, USA).

    Techniques:

    OGD rapidly activates p38 MAPK in rat hippocampal slice cultures. Rat hippocampal slice cultures were exposed to OGD in the presence or absence of the p38 MAPK inhibitor, SB203580 (50 μ m , 2 h prior to OGD). Slices were harvested at 0, 2 and 4

    Journal: The European journal of neuroscience

    Article Title: Increased p38 mitogen-activated protein kinase signaling is involved in the oxidative stress associated with oxygen and glucose deprivation in neonatal hippocampal slice cultures

    doi: 10.1111/j.1460-9568.2011.07786.x

    Figure Lengend Snippet: OGD rapidly activates p38 MAPK in rat hippocampal slice cultures. Rat hippocampal slice cultures were exposed to OGD in the presence or absence of the p38 MAPK inhibitor, SB203580 (50 μ m , 2 h prior to OGD). Slices were harvested at 0, 2 and 4

    Article Snippet: The primary antibodies used were anti-phospho-p38 MAPK and p38 MAPK (Santa Cruz, CA, USA), anti-caspase-3 and anti-cleaved caspase-3 (Cell Signaling, Danvers, MA, USA).

    Techniques:

    OGD increases p38 MAPK-dependent increases in superoxide generation in rat hippocampal slice cultures. Rat hippocampal slice cultures were exposed to OGD in the presence or absence of the p38 MAPK inhibitor, SB203580 (50 μ m , 2 h prior to OGD).

    Journal: The European journal of neuroscience

    Article Title: Increased p38 mitogen-activated protein kinase signaling is involved in the oxidative stress associated with oxygen and glucose deprivation in neonatal hippocampal slice cultures

    doi: 10.1111/j.1460-9568.2011.07786.x

    Figure Lengend Snippet: OGD increases p38 MAPK-dependent increases in superoxide generation in rat hippocampal slice cultures. Rat hippocampal slice cultures were exposed to OGD in the presence or absence of the p38 MAPK inhibitor, SB203580 (50 μ m , 2 h prior to OGD).

    Article Snippet: The primary antibodies used were anti-phospho-p38 MAPK and p38 MAPK (Santa Cruz, CA, USA), anti-caspase-3 and anti-cleaved caspase-3 (Cell Signaling, Danvers, MA, USA).

    Techniques:

    OGD induces regional cell injury in rat hippocampal slice cultures. Rat hippocampal slice cultures were exposed to OGD in the presence or absence of the p38 MAPK inhibitor, SB203580 (50 μ m , 2 h prior to OGD). The effect on cell injury was then

    Journal: The European journal of neuroscience

    Article Title: Increased p38 mitogen-activated protein kinase signaling is involved in the oxidative stress associated with oxygen and glucose deprivation in neonatal hippocampal slice cultures

    doi: 10.1111/j.1460-9568.2011.07786.x

    Figure Lengend Snippet: OGD induces regional cell injury in rat hippocampal slice cultures. Rat hippocampal slice cultures were exposed to OGD in the presence or absence of the p38 MAPK inhibitor, SB203580 (50 μ m , 2 h prior to OGD). The effect on cell injury was then

    Article Snippet: The primary antibodies used were anti-phospho-p38 MAPK and p38 MAPK (Santa Cruz, CA, USA), anti-caspase-3 and anti-cleaved caspase-3 (Cell Signaling, Danvers, MA, USA).

    Techniques:

    OGD increases LDH release in rat hippocampal slice cultures. Rat hippocampal slice cultures were exposed to OGD in the presence or absence of the p38 MAPK inhibitor, SB203580 (50 μ m , 2 h prior to OGD). The time-dependent effect on LDH release

    Journal: The European journal of neuroscience

    Article Title: Increased p38 mitogen-activated protein kinase signaling is involved in the oxidative stress associated with oxygen and glucose deprivation in neonatal hippocampal slice cultures

    doi: 10.1111/j.1460-9568.2011.07786.x

    Figure Lengend Snippet: OGD increases LDH release in rat hippocampal slice cultures. Rat hippocampal slice cultures were exposed to OGD in the presence or absence of the p38 MAPK inhibitor, SB203580 (50 μ m , 2 h prior to OGD). The time-dependent effect on LDH release

    Article Snippet: The primary antibodies used were anti-phospho-p38 MAPK and p38 MAPK (Santa Cruz, CA, USA), anti-caspase-3 and anti-cleaved caspase-3 (Cell Signaling, Danvers, MA, USA).

    Techniques:

    Targeted decreases in p38 MAPK expression in neuronal cells attenuate superoxide generation in rat hippocampal slice cultures. Rat hippocampal slice cultures were transduced with either the AAV-SYN-1-p38 MAPK-AS or the AAV-SYN-1null constructs or were

    Journal: The European journal of neuroscience

    Article Title: Increased p38 mitogen-activated protein kinase signaling is involved in the oxidative stress associated with oxygen and glucose deprivation in neonatal hippocampal slice cultures

    doi: 10.1111/j.1460-9568.2011.07786.x

    Figure Lengend Snippet: Targeted decreases in p38 MAPK expression in neuronal cells attenuate superoxide generation in rat hippocampal slice cultures. Rat hippocampal slice cultures were transduced with either the AAV-SYN-1-p38 MAPK-AS or the AAV-SYN-1null constructs or were

    Article Snippet: The primary antibodies used were anti-phospho-p38 MAPK and p38 MAPK (Santa Cruz, CA, USA), anti-caspase-3 and anti-cleaved caspase-3 (Cell Signaling, Danvers, MA, USA).

    Techniques: Expressing, Transduction, Construct

    OGD increases caspase activation in rat hippocampal slice cultures. Rat hippocampal slice cultures were exposed to OGD in the presence or absence of the p38 MAPK inhibitor, SB203580 (50 μ m , 2 h prior to OGD). Slices were harvested at 0, 4, 8 and

    Journal: The European journal of neuroscience

    Article Title: Increased p38 mitogen-activated protein kinase signaling is involved in the oxidative stress associated with oxygen and glucose deprivation in neonatal hippocampal slice cultures

    doi: 10.1111/j.1460-9568.2011.07786.x

    Figure Lengend Snippet: OGD increases caspase activation in rat hippocampal slice cultures. Rat hippocampal slice cultures were exposed to OGD in the presence or absence of the p38 MAPK inhibitor, SB203580 (50 μ m , 2 h prior to OGD). Slices were harvested at 0, 4, 8 and

    Article Snippet: The primary antibodies used were anti-phospho-p38 MAPK and p38 MAPK (Santa Cruz, CA, USA), anti-caspase-3 and anti-cleaved caspase-3 (Cell Signaling, Danvers, MA, USA).

    Techniques: Activation Assay

    Monostability in the p38 signaling system. Time course of p38 activation in oocytes treated with 300 mM sorbitol. After 4 h of treatment (arrow), oocytes were washed several times with MBS and maintained in MBS without sorbitol. Pools of 20 oocytes were collected at different times to determine pp38, p38, cytochrome c (CC), and cleaved caspase-3 by Western blot. p38 activity is represented as pp38/p38 ratio, given 100% value to the highest activity. Results are represented as mean ± SEM of three independent experiments. A representative Western blot is shown. Caspase-3 activity was determined as reported before. Data are represented as mean ± SEM (n = 3), *p

    Journal: PLoS ONE

    Article Title: Basic Properties of the p38 Signaling Pathway in Response to Hyperosmotic Shock

    doi: 10.1371/journal.pone.0135249

    Figure Lengend Snippet: Monostability in the p38 signaling system. Time course of p38 activation in oocytes treated with 300 mM sorbitol. After 4 h of treatment (arrow), oocytes were washed several times with MBS and maintained in MBS without sorbitol. Pools of 20 oocytes were collected at different times to determine pp38, p38, cytochrome c (CC), and cleaved caspase-3 by Western blot. p38 activity is represented as pp38/p38 ratio, given 100% value to the highest activity. Results are represented as mean ± SEM of three independent experiments. A representative Western blot is shown. Caspase-3 activity was determined as reported before. Data are represented as mean ± SEM (n = 3), *p

    Article Snippet: Membranes were blocked for 1 h with 5% dried skimmed milk in TBST (50 mM Tris, 150 mM NaCl, 100 mM KCl, pH 7.4, and 0.1% Tween 20) and then incubated with the following antibodies: anti-AMPKα (#2532, Cell Signaling), anti-pp38 (Thr180/Tyr182) (#9211, Cell Signaling), anti-p38 (sc-7149, Santa Cruz), and anti-Cytochrome c (556432, BD Pharmingen).

    Techniques: Activation Assay, Western Blot, Activity Assay

    Caspase-3 inhibitor reduces p38 phosphorylation induced by hyperosmotic shock. Oocytes were incubated with 200 mM sorbitol for 4 h in the presence or absence of caspase-3 inhibitor Z-DEVD.fmk (50 μM), or with 400 mM sorbitol for 1 h. Pools of 20 oocytes were collected and pp38, p38, cytochrome c (CC) and AMPK (loading control) were analyzed by Western blot. Caspase-3 activity was determined as the concentration of fluorescent AMC formation from Z-DEVD-AMC substrate, and represented as arbitrary units of caspase-3 activity, giving value 1 to non treated oocytes.

    Journal: PLoS ONE

    Article Title: Basic Properties of the p38 Signaling Pathway in Response to Hyperosmotic Shock

    doi: 10.1371/journal.pone.0135249

    Figure Lengend Snippet: Caspase-3 inhibitor reduces p38 phosphorylation induced by hyperosmotic shock. Oocytes were incubated with 200 mM sorbitol for 4 h in the presence or absence of caspase-3 inhibitor Z-DEVD.fmk (50 μM), or with 400 mM sorbitol for 1 h. Pools of 20 oocytes were collected and pp38, p38, cytochrome c (CC) and AMPK (loading control) were analyzed by Western blot. Caspase-3 activity was determined as the concentration of fluorescent AMC formation from Z-DEVD-AMC substrate, and represented as arbitrary units of caspase-3 activity, giving value 1 to non treated oocytes.

    Article Snippet: Membranes were blocked for 1 h with 5% dried skimmed milk in TBST (50 mM Tris, 150 mM NaCl, 100 mM KCl, pH 7.4, and 0.1% Tween 20) and then incubated with the following antibodies: anti-AMPKα (#2532, Cell Signaling), anti-pp38 (Thr180/Tyr182) (#9211, Cell Signaling), anti-p38 (sc-7149, Santa Cruz), and anti-Cytochrome c (556432, BD Pharmingen).

    Techniques: Incubation, Western Blot, Activity Assay, Concentration Assay

    p38 is ultrasensitive to hyperosmolar sorbitol. Oocytes were treated with increasing concentrations of sorbitol for 4 h and pools of 20 oocytes were collected and lysed to analyze pp38, p38, cytochrome c (CC), and cleaved caspase-3 by Western blot. p38 activity is represented as pp38/p38 ratio, giving 100% value to the highest activity. Results are represented as mean ± SEM of four independent experiments. Hill coefficient (nH) was calculated with SAS 9.2 informatics program and represented with GraphPad Prism 4 program. A representative Western blot is shown. Caspase-3 activity was determined at different concentrations of sorbitol as reported before. Data are represented as mean ± SEM (n = 3), *p

    Journal: PLoS ONE

    Article Title: Basic Properties of the p38 Signaling Pathway in Response to Hyperosmotic Shock

    doi: 10.1371/journal.pone.0135249

    Figure Lengend Snippet: p38 is ultrasensitive to hyperosmolar sorbitol. Oocytes were treated with increasing concentrations of sorbitol for 4 h and pools of 20 oocytes were collected and lysed to analyze pp38, p38, cytochrome c (CC), and cleaved caspase-3 by Western blot. p38 activity is represented as pp38/p38 ratio, giving 100% value to the highest activity. Results are represented as mean ± SEM of four independent experiments. Hill coefficient (nH) was calculated with SAS 9.2 informatics program and represented with GraphPad Prism 4 program. A representative Western blot is shown. Caspase-3 activity was determined at different concentrations of sorbitol as reported before. Data are represented as mean ± SEM (n = 3), *p

    Article Snippet: Membranes were blocked for 1 h with 5% dried skimmed milk in TBST (50 mM Tris, 150 mM NaCl, 100 mM KCl, pH 7.4, and 0.1% Tween 20) and then incubated with the following antibodies: anti-AMPKα (#2532, Cell Signaling), anti-pp38 (Thr180/Tyr182) (#9211, Cell Signaling), anti-p38 (sc-7149, Santa Cruz), and anti-Cytochrome c (556432, BD Pharmingen).

    Techniques: Western Blot, Activity Assay

    Microinjection of cytochrome c (CC) induces caspase-3 activation and p38 phosphorylation. (A) Caspase inhibitors reduce p38 phosphorylation induced by cytochrome c injection. Oocytes were injected with MBS, horse cytochrome c (CC) (0.5 μM final concentration), CC (0.5 μM) plus Z-VAD.fmk (5 μM), CC (0.5 μM) plus Ac-DEVD-CHO (100 nM), or non injected (control). Pools of 20 oocytes were collected at different times and caspase-3 activity was determined as the concentration of fluorescent AMC formation from Z-DEVD-AMC substrate, and represented as arbitrary units of caspase-3 activity, giving value 1 to MBS injected oocytes. pp38, p38 and AMPK (loading control) were analyzed by Western blot. The result presented is representative of three independent experiments. (B) Injection of BSA or cytochrome c from Saccharomyces cerevisiae (CC Yeast) does not induce caspase-3 activation and p38 phosphorylation. Oocytes were injected with MBS, horse cytochrome c (CC) (0.5 μM), yeast cytochrome c (CC Yeast) (0.5 μM), BSA (0.5 μM), or non injected (control). Pools of 20 oocytes were collected at different times and caspase-3 activity was determined as described before. pp38, p38 and AMPK were analyzed by Western blot. (C) DMSO injection does not interfere with p38 phosphorylation and caspase-3 activation induced by cytochrome c. Oocytes were injected with MBS plus DMSO (diluted 1:50 in MBS), horse cytochrome c (CC) (0.5 μM) plus DMSO (diluted 1:50 in CC), CC (0.5 μM) plus Z-VAD.fmk (50 μM), or CC (0.5 μM) plus Ac-DEVD-CHO (1 μM). Pools of 20 oocytes were collected at different times and caspase-3 activity was determined as described before. pp38, p38 and AMPK were analyzed by Western blot.

    Journal: PLoS ONE

    Article Title: Basic Properties of the p38 Signaling Pathway in Response to Hyperosmotic Shock

    doi: 10.1371/journal.pone.0135249

    Figure Lengend Snippet: Microinjection of cytochrome c (CC) induces caspase-3 activation and p38 phosphorylation. (A) Caspase inhibitors reduce p38 phosphorylation induced by cytochrome c injection. Oocytes were injected with MBS, horse cytochrome c (CC) (0.5 μM final concentration), CC (0.5 μM) plus Z-VAD.fmk (5 μM), CC (0.5 μM) plus Ac-DEVD-CHO (100 nM), or non injected (control). Pools of 20 oocytes were collected at different times and caspase-3 activity was determined as the concentration of fluorescent AMC formation from Z-DEVD-AMC substrate, and represented as arbitrary units of caspase-3 activity, giving value 1 to MBS injected oocytes. pp38, p38 and AMPK (loading control) were analyzed by Western blot. The result presented is representative of three independent experiments. (B) Injection of BSA or cytochrome c from Saccharomyces cerevisiae (CC Yeast) does not induce caspase-3 activation and p38 phosphorylation. Oocytes were injected with MBS, horse cytochrome c (CC) (0.5 μM), yeast cytochrome c (CC Yeast) (0.5 μM), BSA (0.5 μM), or non injected (control). Pools of 20 oocytes were collected at different times and caspase-3 activity was determined as described before. pp38, p38 and AMPK were analyzed by Western blot. (C) DMSO injection does not interfere with p38 phosphorylation and caspase-3 activation induced by cytochrome c. Oocytes were injected with MBS plus DMSO (diluted 1:50 in MBS), horse cytochrome c (CC) (0.5 μM) plus DMSO (diluted 1:50 in CC), CC (0.5 μM) plus Z-VAD.fmk (50 μM), or CC (0.5 μM) plus Ac-DEVD-CHO (1 μM). Pools of 20 oocytes were collected at different times and caspase-3 activity was determined as described before. pp38, p38 and AMPK were analyzed by Western blot.

    Article Snippet: Membranes were blocked for 1 h with 5% dried skimmed milk in TBST (50 mM Tris, 150 mM NaCl, 100 mM KCl, pH 7.4, and 0.1% Tween 20) and then incubated with the following antibodies: anti-AMPKα (#2532, Cell Signaling), anti-pp38 (Thr180/Tyr182) (#9211, Cell Signaling), anti-p38 (sc-7149, Santa Cruz), and anti-Cytochrome c (556432, BD Pharmingen).

    Techniques: Activation Assay, Injection, Concentration Assay, Activity Assay, Western Blot

    Hyperosmotic shock activates p38 in Xenopus laevis oocytes. Oocytes (stage VI) were treated with sorbitol (400 mM) and pools of 20 oocytes were lysed at different times to analyze pp38, p38, cytochrome c (CC), and cleaved caspase-3 by Western blot. p38 activity is represented as pp38/p38 ratio, giving 100% value to the highest activity. Results are represented as mean ± SEM of four independent experiments. A representative Western blot is shown. Caspase-3 activity was determined at different times, using the synthetic peptide Z-DEVD-AMC as a substrate and giving value 1 to non treated oocytes. Data are represented as mean ± SEM (n = 3), *p

    Journal: PLoS ONE

    Article Title: Basic Properties of the p38 Signaling Pathway in Response to Hyperosmotic Shock

    doi: 10.1371/journal.pone.0135249

    Figure Lengend Snippet: Hyperosmotic shock activates p38 in Xenopus laevis oocytes. Oocytes (stage VI) were treated with sorbitol (400 mM) and pools of 20 oocytes were lysed at different times to analyze pp38, p38, cytochrome c (CC), and cleaved caspase-3 by Western blot. p38 activity is represented as pp38/p38 ratio, giving 100% value to the highest activity. Results are represented as mean ± SEM of four independent experiments. A representative Western blot is shown. Caspase-3 activity was determined at different times, using the synthetic peptide Z-DEVD-AMC as a substrate and giving value 1 to non treated oocytes. Data are represented as mean ± SEM (n = 3), *p

    Article Snippet: Membranes were blocked for 1 h with 5% dried skimmed milk in TBST (50 mM Tris, 150 mM NaCl, 100 mM KCl, pH 7.4, and 0.1% Tween 20) and then incubated with the following antibodies: anti-AMPKα (#2532, Cell Signaling), anti-pp38 (Thr180/Tyr182) (#9211, Cell Signaling), anti-p38 (sc-7149, Santa Cruz), and anti-Cytochrome c (556432, BD Pharmingen).

    Techniques: Western Blot, Activity Assay

    Bimodal and time-dependent response of the p38 system to hyperosmolar sorbitol. (A) Oocytes were incubated with 200 mM sorbitol for 2 h and pp38, p38, and cytochrome c (CC) were measured by Western blot in individual oocytes. p38 activity is represented as pp38/p38 ratio, taking as maximum activity (100% value) the oocytes treated with 400 mM sorbitol (M). Each box represents one individual oocyte. Results are the pool of three independent experiments. The Western blot shows a representative experiment. The arrow indicates no correlation between p38 activation and cytochrome c release at 2 h. (B) p38 activity in individual oocytes treated with 200 mM sorbitol for 4 h. Activity was measured by Western blot, as described previously, and the results expressed as pp38/p38, taking as maximum activity the value obtained with 400 mM sorbitol. (C) Correlation between pp38 and cytochrome c in oocytes incubated with 200 mM sorbitol for 2 h. pp38 and cytochrome c (CC) levels were determined by Western blot in individual oocytes (n = 50) from 5 independent experiments, giving 100% value to individual oocytes treated with 400 mM sorbitol. The data obtained for each oocyte was represented graphically and the correlation coefficient (Pearson r, Spearman r) calculated with GraphPad Prism4. (D) Correlation between pp38 and cytochrome c in oocytes incubated with 200 mM sorbitol for 4 h. pp38 and cytochrome c (CC) were determined by Western blot in individual oocytes (n = 50) as reported before, the data obtained represented graphically, and the correlation coefficients calculated with GraphPad Prism4.

    Journal: PLoS ONE

    Article Title: Basic Properties of the p38 Signaling Pathway in Response to Hyperosmotic Shock

    doi: 10.1371/journal.pone.0135249

    Figure Lengend Snippet: Bimodal and time-dependent response of the p38 system to hyperosmolar sorbitol. (A) Oocytes were incubated with 200 mM sorbitol for 2 h and pp38, p38, and cytochrome c (CC) were measured by Western blot in individual oocytes. p38 activity is represented as pp38/p38 ratio, taking as maximum activity (100% value) the oocytes treated with 400 mM sorbitol (M). Each box represents one individual oocyte. Results are the pool of three independent experiments. The Western blot shows a representative experiment. The arrow indicates no correlation between p38 activation and cytochrome c release at 2 h. (B) p38 activity in individual oocytes treated with 200 mM sorbitol for 4 h. Activity was measured by Western blot, as described previously, and the results expressed as pp38/p38, taking as maximum activity the value obtained with 400 mM sorbitol. (C) Correlation between pp38 and cytochrome c in oocytes incubated with 200 mM sorbitol for 2 h. pp38 and cytochrome c (CC) levels were determined by Western blot in individual oocytes (n = 50) from 5 independent experiments, giving 100% value to individual oocytes treated with 400 mM sorbitol. The data obtained for each oocyte was represented graphically and the correlation coefficient (Pearson r, Spearman r) calculated with GraphPad Prism4. (D) Correlation between pp38 and cytochrome c in oocytes incubated with 200 mM sorbitol for 4 h. pp38 and cytochrome c (CC) were determined by Western blot in individual oocytes (n = 50) as reported before, the data obtained represented graphically, and the correlation coefficients calculated with GraphPad Prism4.

    Article Snippet: Membranes were blocked for 1 h with 5% dried skimmed milk in TBST (50 mM Tris, 150 mM NaCl, 100 mM KCl, pH 7.4, and 0.1% Tween 20) and then incubated with the following antibodies: anti-AMPKα (#2532, Cell Signaling), anti-pp38 (Thr180/Tyr182) (#9211, Cell Signaling), anti-p38 (sc-7149, Santa Cruz), and anti-Cytochrome c (556432, BD Pharmingen).

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