p-p38 Search Results


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  • 99
    Cell Signaling Technology Inc p p38
    p38 is involved in CXCL13-induced pain hypersensitivity. ( A ) Western blot shows that <t>pp38</t> expression in the DRG was increased 3 days after CFA in WT mice, but not in Cxcr5 KO mice. *P
    P P38, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 8343 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti p p38
    p38 is involved in CXCL13-induced pain hypersensitivity. ( A ) Western blot shows that <t>pp38</t> expression in the DRG was increased 3 days after CFA in WT mice, but not in Cxcr5 KO mice. *P
    Anti P P38, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1537 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Cell Signaling Technology Inc p p38 mapk
    LDIR inhibits cell growth and protein synthesis and induces bystander effects. Cell signaling pathways affected by LDIR. LDIR downregulates c-Myc and upregulates p21 WAF1/CIP1 via stimulation of TGFβ and PP2A. PP2A also inhibits mTOR signaling with repression of S6K activation and 4EBP1 phosphorylation that resulted in decrease in protein synthesis. Furthermore, LDIR induces bystander effects through <t>p38</t> <t>MAPK</t> activation. Directly-irradiated cells release cytokine signals that affect non-irradiated (bystander) cells.
    P P38 Mapk, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 1384 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology p p38
    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 <t>p-p38</t> 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.
    P P38, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 1509 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    p p38  (Abcam)
    99
    Abcam p p38
    lncRNA-ROR mediated myocardial hypoxia/reoxygenation (H/R) by regulating the <t>p38/MAPK</t> pathway. H9c2 cells and human cardiomyocytes (HCM) were transfected with lncRNA-ROR overexpression vector (lncRNA-ROR) and inhibition vector (si-lncRNA-ROR). After H/R treatment for 24 h, ( A ) the protein levels of ERK and p38 were examined by western blot and ( B ) the mRNA expressions of ERK and p38 were determined by qRT-PCR. Data are reported as means±SE. *P
    P P38, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 614 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc rabbit anti p p38
    lncRNA-ROR mediated myocardial hypoxia/reoxygenation (H/R) by regulating the <t>p38/MAPK</t> pathway. H9c2 cells and human cardiomyocytes (HCM) were transfected with lncRNA-ROR overexpression vector (lncRNA-ROR) and inhibition vector (si-lncRNA-ROR). After H/R treatment for 24 h, ( A ) the protein levels of ERK and p38 were examined by western blot and ( B ) the mRNA expressions of ERK and p38 were determined by qRT-PCR. Data are reported as means±SE. *P
    Rabbit Anti P P38, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 406 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti p p38 mapk
    SGE regulates AKT and <t>p38</t> <t>MAPK</t> phosphorylation at the beginning of myogenesis. C2C12 cells were cultured in GM. To begin the differentiation, this medium was withdrawn and then replaced by DM. Treatment was started by adding 10 nM of SGE or 1 nM of synthetic 1α,25(|OH) 2 D 3 (1,25D) in DM during different periods of time (1–48 h). Western blots were performed with anti p-AKT, total AKT, p-p38 MAPK, and p38α and tubulin antibodies. Tubulin was used as loading protein marker. Representative blots from three independent experiments (upper panel) and their quantification (lower panel) are shown. Protein bands quantification was done using ImageJ. Results were then represented in bar graphs as phosphorylated protein normalized with its corresponding total from treated conditions and referred to control (Fold) (lower panel). Significant differences between conditions at each time point were analyzed by Student’s t -test, ** P
    Anti P P38 Mapk, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 401 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Santa Cruz Biotechnology anti p p38
    SGE regulates AKT and <t>p38</t> <t>MAPK</t> phosphorylation at the beginning of myogenesis. C2C12 cells were cultured in GM. To begin the differentiation, this medium was withdrawn and then replaced by DM. Treatment was started by adding 10 nM of SGE or 1 nM of synthetic 1α,25(|OH) 2 D 3 (1,25D) in DM during different periods of time (1–48 h). Western blots were performed with anti p-AKT, total AKT, p-p38 MAPK, and p38α and tubulin antibodies. Tubulin was used as loading protein marker. Representative blots from three independent experiments (upper panel) and their quantification (lower panel) are shown. Protein bands quantification was done using ImageJ. Results were then represented in bar graphs as phosphorylated protein normalized with its corresponding total from treated conditions and referred to control (Fold) (lower panel). Significant differences between conditions at each time point were analyzed by Student’s t -test, ** P
    Anti P P38, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 346 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti pp38
    (IVIg + LPS)-induced MAPK phosphorylation is lower in monocytes from people with the FcγRIIA risk variant. (A) Monocytes from healthy participants with the non-risk genotype (CC) or the risk genotype (TT) were unstimulated or stimulated with LPS (100 ng/ml), IVIg (5 mg/ml), or both, for 0, 10, 40, or 120 min. Cell lysates (2.5 × 10 5 cells / time point) were prepared at the indicated times. Lysates were separated by SDS-PAGE and analyzed by western blotting using phosphospecific antibodies for ERK1/2, p38, and using GAPDH, as a loading control. Representative western blot from participants with the TT genotype are shown in (A) . Results are representative of n = 3 experiments per genotype; monocytes were derived from 1 participant for each of 3 independent experiments. Densitometry for pERK1/2 and <t>pp38;</t> normalized to GAPDH and relative to LPS 10 min; are averaged from n = 3 independent experiments and shown below each band and values are graphed as mean ± SEM for each genotype. Monocytes were pre-treated for 1 h with an appropriate volume of DMSO, as a vehicle control, or (B) the ERK1/2 inhibitors, PD98059 (PD) and SCH772984 (SCH); or (C) the p38 inhibitors, SB203580 (SB) and BIRB796 (BIR) and were unstimulated (C) or stimulated with LPS (100 ng/ml), IVIg (5 mg/ml), or both for 24 h. Clarified cell supernatants were analyzed for IL-10 by ELISA. (B,C) Values are reported as mean ± SEM for n = 7 participants for the CC genotype and n = 4 participants for the TT genotype performed as independent experiments, and assayed in duplicate. * p
    Anti Pp38, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 274 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Cell Signaling Technology Inc p p38 t180 y182
    (IVIg + LPS)-induced MAPK phosphorylation is lower in monocytes from people with the FcγRIIA risk variant. (A) Monocytes from healthy participants with the non-risk genotype (CC) or the risk genotype (TT) were unstimulated or stimulated with LPS (100 ng/ml), IVIg (5 mg/ml), or both, for 0, 10, 40, or 120 min. Cell lysates (2.5 × 10 5 cells / time point) were prepared at the indicated times. Lysates were separated by SDS-PAGE and analyzed by western blotting using phosphospecific antibodies for ERK1/2, p38, and using GAPDH, as a loading control. Representative western blot from participants with the TT genotype are shown in (A) . Results are representative of n = 3 experiments per genotype; monocytes were derived from 1 participant for each of 3 independent experiments. Densitometry for pERK1/2 and <t>pp38;</t> normalized to GAPDH and relative to LPS 10 min; are averaged from n = 3 independent experiments and shown below each band and values are graphed as mean ± SEM for each genotype. Monocytes were pre-treated for 1 h with an appropriate volume of DMSO, as a vehicle control, or (B) the ERK1/2 inhibitors, PD98059 (PD) and SCH772984 (SCH); or (C) the p38 inhibitors, SB203580 (SB) and BIRB796 (BIR) and were unstimulated (C) or stimulated with LPS (100 ng/ml), IVIg (5 mg/ml), or both for 24 h. Clarified cell supernatants were analyzed for IL-10 by ELISA. (B,C) Values are reported as mean ± SEM for n = 7 participants for the CC genotype and n = 4 participants for the TT genotype performed as independent experiments, and assayed in duplicate. * p
    P P38 T180 Y182, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 88/100, based on 45 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Becton Dickinson p p38
    Phosphorylation of the MAPKs <t>p38,</t> ERK1/2 and JNK is increased upon IV/ S. aureus super-infection. Calu-3 ( a – d ) or A549 ( e – i ) cells were infected with IV H1N1(M) ( a – h ) or H3N2 ( i ) (MOI 5) for 0.5 h and super-infected with S. aureus 6850 ( a – i ), SH1000 or MRSA USA300 ( i ) (MOI 50). Extracellular bacteria were removed by gentamicin treatment 3 h after bacterial infection. At 8 h p.i. cells were lysed and whole cell lysates were subjected to Western blot analysis monitoring P-p38, P-ERK1/2, P-JNK, PGN and PB1. Equal protein loading was verified by detection of p38, ERK2 and JNK ( a , e , i ). Original blots are shown in Supplementary Fig. S8b (upper panel: Fig. 5a, lower panel: Fig. 5e) and S8c (upper panel: Fig. 5i [S. aureus 6850], lower panel [ S. aureus SH1000, USA300]). Relative levels of P-p38, P-ERK1/2 and P-JNK compared to p38, ERK2 or JNK bands, respectively, were quantified by using ImageJ 2006.02.01 software ( b – d , f – h ). Means ± SD of at least three independent experiments are shown. Two-tailed unpaired t-tests were performed for comparison of IV H1N1(M)-infected and IV H1N1(M)/ S. aureus 6850 super-infected samples (*p
    P P38, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 129 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Cell Signaling Technology Inc p p38 mapk thr180 tyr182
    Phosphorylation of the MAPKs <t>p38,</t> ERK1/2 and JNK is increased upon IV/ S. aureus super-infection. Calu-3 ( a – d ) or A549 ( e – i ) cells were infected with IV H1N1(M) ( a – h ) or H3N2 ( i ) (MOI 5) for 0.5 h and super-infected with S. aureus 6850 ( a – i ), SH1000 or MRSA USA300 ( i ) (MOI 50). Extracellular bacteria were removed by gentamicin treatment 3 h after bacterial infection. At 8 h p.i. cells were lysed and whole cell lysates were subjected to Western blot analysis monitoring P-p38, P-ERK1/2, P-JNK, PGN and PB1. Equal protein loading was verified by detection of p38, ERK2 and JNK ( a , e , i ). Original blots are shown in Supplementary Fig. S8b (upper panel: Fig. 5a, lower panel: Fig. 5e) and S8c (upper panel: Fig. 5i [S. aureus 6850], lower panel [ S. aureus SH1000, USA300]). Relative levels of P-p38, P-ERK1/2 and P-JNK compared to p38, ERK2 or JNK bands, respectively, were quantified by using ImageJ 2006.02.01 software ( b – d , f – h ). Means ± SD of at least three independent experiments are shown. Two-tailed unpaired t-tests were performed for comparison of IV H1N1(M)-infected and IV H1N1(M)/ S. aureus 6850 super-infected samples (*p
    P P38 Mapk Thr180 Tyr182, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 67 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology p p38 mapk
    Effect of TPA on fisetin-induced inhibition of uPA expression, cell migration and invasion in SiHa cells. Cells were pretreated with or without fisetin for 2 h, and then treated with or without 50 ng/ml of TPA for 24 h. (A) The phosphorylated protein and total protein of <t>p38</t> <t>MAPK</t> were determined by Western blotting. (B) The mRNA level of uPA after each treatment was examined by RT-PCR. (C) The protein level of uPA was determined by Western blotting. β-actin was used as the internal control. (D) The conditioned medium from each treatment was collected, and the uPA activity was determined by casein zymography. The migratory (E) and invasive (F) ability of SiHa cells after treatment were determined. Migrating and invading cells were photographed using phase-contrast microscopy. Values represent mean ± SD of three independent experiments. *, P
    P P38 Mapk, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 153 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Beyotime p p38
    Schematic representation of proposed signaling pathways suggested by the results of this study. Methylglyoxal increases production of reactive oxygen species (ROS), which then reduces the mitochondrial membrane potential (MMP) and ATP production via UCP2 upregulation. UCP2 upregulation in turn leads to β -cell damage and, ultimately, impairment of insulin secretion. ROS also may induce β -cell damage and impair insulin secretion directly via upregulation of MAPKs <t>(JNK/P38).</t>
    P P38, supplied by Beyotime, used in various techniques. Bioz Stars score: 92/100, based on 60 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    p38 is involved in CXCL13-induced pain hypersensitivity. ( A ) Western blot shows that pp38 expression in the DRG was increased 3 days after CFA in WT mice, but not in Cxcr5 KO mice. *P

    Journal: Scientific Reports

    Article Title: CXCL13/CXCR5 enhances sodium channel Nav1.8 current density via p38 MAP kinase in primary sensory neurons following inflammatory pain

    doi: 10.1038/srep34836

    Figure Lengend Snippet: p38 is involved in CXCL13-induced pain hypersensitivity. ( A ) Western blot shows that pp38 expression in the DRG was increased 3 days after CFA in WT mice, but not in Cxcr5 KO mice. *P

    Article Snippet: The blots were blocked with 5% milk and incubated overnight at 4 °C with antibody against CXCL13 (Goat, 1:100, Santa Cruz, sc-8182), CXCR5 (rabbit, 1:100, Santa Cruz, sc-30029), pp38 (rabbit, 1:1000, Cell Signaling, #9215), p38 (rabbit, 1:1000, Cell Signaling, #9212), and GAPDH (mouse, 1:20000, Millipore, MAB374).

    Techniques: Western Blot, Expressing, Mouse Assay

    Clorgyline and phenelzine, inhibitors of MAO-A, can overcome Enz resistance. a The in vivo PDX-PCa mouse model data revealed that injection with Enz (30 mg/kg/every other day) increased the MAO-A, ARv7, and p-p38 level. b , c Mice ( n = 10) implanted with EnzR3-22Rv1 xenografts were treated with vehicle control, Enz (30 mg/kg), clorgyline (10 mg/kg), phenelzine (30 mg/kg), Enz+clorgyline (30 mg/kg + 10 mg/kg), or Enz+phenelzine (30 mg/kg + 30 mg/kg). After sacrifice, tumors of the six groups were collected and weighed. d , e Mice ( n = 6) implanted with EnzR1-C4-2 xenografts received the same treatments as EnzR3-22Rv1. After sacrifice, tumors of the six groups were collected and weighed. f IHC staining of Ki67 and VEGF-A in EnzR3-22Rv1 tumors were performed; (scale bar = 50 μm). g IHC staining of Ki67 and VEGF-A in EnzR1-C4-2 tumors were performed; (scale bar = 50 μm). Data represent the mean ± SEM, error bars represent SEM. p -value was determined by two-tailed paired t -test.

    Journal: Nature Communications

    Article Title: The MAO inhibitors phenelzine and clorgyline revert enzalutamide resistance in castration resistant prostate cancer

    doi: 10.1038/s41467-020-15396-5

    Figure Lengend Snippet: Clorgyline and phenelzine, inhibitors of MAO-A, can overcome Enz resistance. a The in vivo PDX-PCa mouse model data revealed that injection with Enz (30 mg/kg/every other day) increased the MAO-A, ARv7, and p-p38 level. b , c Mice ( n = 10) implanted with EnzR3-22Rv1 xenografts were treated with vehicle control, Enz (30 mg/kg), clorgyline (10 mg/kg), phenelzine (30 mg/kg), Enz+clorgyline (30 mg/kg + 10 mg/kg), or Enz+phenelzine (30 mg/kg + 30 mg/kg). After sacrifice, tumors of the six groups were collected and weighed. d , e Mice ( n = 6) implanted with EnzR1-C4-2 xenografts received the same treatments as EnzR3-22Rv1. After sacrifice, tumors of the six groups were collected and weighed. f IHC staining of Ki67 and VEGF-A in EnzR3-22Rv1 tumors were performed; (scale bar = 50 μm). g IHC staining of Ki67 and VEGF-A in EnzR1-C4-2 tumors were performed; (scale bar = 50 μm). Data represent the mean ± SEM, error bars represent SEM. p -value was determined by two-tailed paired t -test.

    Article Snippet: The p-p38 antibody (#9211) was purchased from Cell signaling Technology (Danvers, MA).

    Techniques: In Vivo, Injection, Mouse Assay, Immunohistochemistry, Staining, Two Tailed Test

    Enz upregulates the MAO-A transactivation in transcriptional level and protein level. a Schematic depiction of putative ARE on MAO-A promoter region. The mutant ARE was marked by italic font. b ChIP assay was performed to identify that endogenous AR and Flag-ARv7 bind to the putative ARE on MAO-A promoter in EnzR1-C4-2 and EnzS1-pWPI-flag-ARv7 cells. c EnzR1 cells were infected by Flag-ARv7 or Flag-ARfl virus. The ChIP assay was performed to analyze Flag-AR and Flag-ARv7 binding on MAO-A promoter region (left) and PSA promoter region (right) in EnzR1 cells; ( n = 3 biological independent samples). d PC3 pWPI and pWPI-ARV7 cells were transfected by the PGL3-MAO-A promoter plasmid, and the promoter activity was determined by luciferase assay; ( n = 3 biological independent samples). e EnzS1-C4-2 cells were treated with Enz at different time points and the MAO-A promoter activity was identified by luciferase reporter assay; ( n = 3 biological independent samples). f MAO-A promoter with mutated ARE was constructed into PGL3 plasmid, and then the MAO-A promoter activity was analyzed in PC3 and PC3-oeARv7 cells; ( n = 3 biological independent samples). g ARv7 and ARfl were transfected into EnzS1-C4-2 cells. And then the PGL3-MAO-A promoter luciferase activity was assayed; ( n = 3 biological independent samples). h ARv7 and ARfl were transfected into EnzS1-C4-2 cells. And then the MAO-A expression was analyzed by western blot (WB). i EnzS1-C4-2 and EnzR1-C4-2 cells were treated with CHX for different time points. The MAO-A protein level was analyzed by WB. j EnzS1-C4-2 and EnzR1-C4-2 cells were treated with MG-132. The MAO-A protein level was analyzed by WB. k MAO-A phosphorylation is increased in EnzR1-C4-2 cells and the lysine phosphorylation of MAO-A was detected by specific antibody. l The phosphorylation of p38 (p-p38) protein levels were detected by WB in EnzS1-C4-2 and EnzR1-C4-2 cells. m EnzS1-C4-2 and EnzR1-C4-2 cells were treated with different concentrations of p38 inhibitor IV (0, 0.5, 1, and 2 μM) for 48 h. The MAO-A protein levels were analyzed by WB. n EnzR1-C4-2 cells were treated with p38 inhibitors for 48 h, and then the MAO-A protein was pulled down, MAO-A p-Ser level was examined by WB. For c and d , data represent the mean ± SEM, error bars represent SEM. p -value was determined by two-tailed paired t -test.

    Journal: Nature Communications

    Article Title: The MAO inhibitors phenelzine and clorgyline revert enzalutamide resistance in castration resistant prostate cancer

    doi: 10.1038/s41467-020-15396-5

    Figure Lengend Snippet: Enz upregulates the MAO-A transactivation in transcriptional level and protein level. a Schematic depiction of putative ARE on MAO-A promoter region. The mutant ARE was marked by italic font. b ChIP assay was performed to identify that endogenous AR and Flag-ARv7 bind to the putative ARE on MAO-A promoter in EnzR1-C4-2 and EnzS1-pWPI-flag-ARv7 cells. c EnzR1 cells were infected by Flag-ARv7 or Flag-ARfl virus. The ChIP assay was performed to analyze Flag-AR and Flag-ARv7 binding on MAO-A promoter region (left) and PSA promoter region (right) in EnzR1 cells; ( n = 3 biological independent samples). d PC3 pWPI and pWPI-ARV7 cells were transfected by the PGL3-MAO-A promoter plasmid, and the promoter activity was determined by luciferase assay; ( n = 3 biological independent samples). e EnzS1-C4-2 cells were treated with Enz at different time points and the MAO-A promoter activity was identified by luciferase reporter assay; ( n = 3 biological independent samples). f MAO-A promoter with mutated ARE was constructed into PGL3 plasmid, and then the MAO-A promoter activity was analyzed in PC3 and PC3-oeARv7 cells; ( n = 3 biological independent samples). g ARv7 and ARfl were transfected into EnzS1-C4-2 cells. And then the PGL3-MAO-A promoter luciferase activity was assayed; ( n = 3 biological independent samples). h ARv7 and ARfl were transfected into EnzS1-C4-2 cells. And then the MAO-A expression was analyzed by western blot (WB). i EnzS1-C4-2 and EnzR1-C4-2 cells were treated with CHX for different time points. The MAO-A protein level was analyzed by WB. j EnzS1-C4-2 and EnzR1-C4-2 cells were treated with MG-132. The MAO-A protein level was analyzed by WB. k MAO-A phosphorylation is increased in EnzR1-C4-2 cells and the lysine phosphorylation of MAO-A was detected by specific antibody. l The phosphorylation of p38 (p-p38) protein levels were detected by WB in EnzS1-C4-2 and EnzR1-C4-2 cells. m EnzS1-C4-2 and EnzR1-C4-2 cells were treated with different concentrations of p38 inhibitor IV (0, 0.5, 1, and 2 μM) for 48 h. The MAO-A protein levels were analyzed by WB. n EnzR1-C4-2 cells were treated with p38 inhibitors for 48 h, and then the MAO-A protein was pulled down, MAO-A p-Ser level was examined by WB. For c and d , data represent the mean ± SEM, error bars represent SEM. p -value was determined by two-tailed paired t -test.

    Article Snippet: The p-p38 antibody (#9211) was purchased from Cell signaling Technology (Danvers, MA).

    Techniques: Mutagenesis, Chromatin Immunoprecipitation, Infection, Binding Assay, Transfection, Plasmid Preparation, Activity Assay, Luciferase, Reporter Assay, Construct, Expressing, Western Blot, Two Tailed Test

    Effects of MAPK inhibitors on the levels of LPS-induced nitrite accumulation and iNOS expression. Cocultured cells were treated for 24 µM of SB203580 (a p38 MAPK inhibitor) or PD98059 (an ERK MAPK inhibitor), which were simultaneously with LPS (100 ng/ml). SB203580 (A) and PD98059 (B) significantly suppressed the LPS-induced accumulation of nitrite and expression of iNOS (C). The data are represented as mean ± SEM based on 3–5 independent experiments. * p

    Journal: PLoS ONE

    Article Title: L-Ascorbate Attenuates the Endotoxin-Induced Production of Inflammatory Mediators by Inhibiting MAPK Activation and NF-?B Translocation in Cortical Neurons/Glia Cocultures

    doi: 10.1371/journal.pone.0097276

    Figure Lengend Snippet: Effects of MAPK inhibitors on the levels of LPS-induced nitrite accumulation and iNOS expression. Cocultured cells were treated for 24 µM of SB203580 (a p38 MAPK inhibitor) or PD98059 (an ERK MAPK inhibitor), which were simultaneously with LPS (100 ng/ml). SB203580 (A) and PD98059 (B) significantly suppressed the LPS-induced accumulation of nitrite and expression of iNOS (C). The data are represented as mean ± SEM based on 3–5 independent experiments. * p

    Article Snippet: Antibodies against p38, phospho-p38 (p-p38), ERK, phospho-ERK (p-ERK), and IκB-α were purchased from Cell Signaling Technology (Beverly, MA, USA).

    Techniques: Expressing

    MAPK Inhibitors and Vit. C inhibited the LPS-induced phosphorylation of p38 and ERK. Cells were treated for 180(control), LPS (100 ng/ml) alone, or LPS combined with 20 µM of the p38 inhibitor SB203580 or ERK inhibitor PD98059, or 10 mM of Vit. C, and then harvested to conduct western blot analyses of p-p38 (A) and p-ERK (p-p42/44) (B). The data are represented as the mean ± SEM based on 3–4 independent experiments. ** p

    Journal: PLoS ONE

    Article Title: L-Ascorbate Attenuates the Endotoxin-Induced Production of Inflammatory Mediators by Inhibiting MAPK Activation and NF-?B Translocation in Cortical Neurons/Glia Cocultures

    doi: 10.1371/journal.pone.0097276

    Figure Lengend Snippet: MAPK Inhibitors and Vit. C inhibited the LPS-induced phosphorylation of p38 and ERK. Cells were treated for 180(control), LPS (100 ng/ml) alone, or LPS combined with 20 µM of the p38 inhibitor SB203580 or ERK inhibitor PD98059, or 10 mM of Vit. C, and then harvested to conduct western blot analyses of p-p38 (A) and p-ERK (p-p42/44) (B). The data are represented as the mean ± SEM based on 3–4 independent experiments. ** p

    Article Snippet: Antibodies against p38, phospho-p38 (p-p38), ERK, phospho-ERK (p-ERK), and IκB-α were purchased from Cell Signaling Technology (Beverly, MA, USA).

    Techniques: Western Blot

    Time course of the LPS-stimulated phosphorylation of p38 (p-p38) and ERK (p-ERK). Cocultured cells were treated with LPS (100 ng/ml) for the indicated times (30, 60, 120, 180, and 300 min). The cells were harvested to analyze the levels of p-p38 (A) and p-ERK (p-p42/44) (B) protein expression by using western blotting. LPS stimulated the phosphorylation of both p38 and ERK, but the phosphorylation of p38 appeared at 60-min post LPS, whereas that of ERK appeared substantially later at 180 min. The data are represented as mean ± SEM based on 3–4 independent experiments. * p

    Journal: PLoS ONE

    Article Title: L-Ascorbate Attenuates the Endotoxin-Induced Production of Inflammatory Mediators by Inhibiting MAPK Activation and NF-?B Translocation in Cortical Neurons/Glia Cocultures

    doi: 10.1371/journal.pone.0097276

    Figure Lengend Snippet: Time course of the LPS-stimulated phosphorylation of p38 (p-p38) and ERK (p-ERK). Cocultured cells were treated with LPS (100 ng/ml) for the indicated times (30, 60, 120, 180, and 300 min). The cells were harvested to analyze the levels of p-p38 (A) and p-ERK (p-p42/44) (B) protein expression by using western blotting. LPS stimulated the phosphorylation of both p38 and ERK, but the phosphorylation of p38 appeared at 60-min post LPS, whereas that of ERK appeared substantially later at 180 min. The data are represented as mean ± SEM based on 3–4 independent experiments. * p

    Article Snippet: Antibodies against p38, phospho-p38 (p-p38), ERK, phospho-ERK (p-ERK), and IκB-α were purchased from Cell Signaling Technology (Beverly, MA, USA).

    Techniques: Expressing, Western Blot

    Effects of MAPK inhibitors on LPS-induced IL-6 and MIP-2 production. Cocultured cells were treated with 20 µM of the p38 inhibitor SB203580 or the ERK inhibitor PD98059, which were simultaneously added with LPS (100 ng/ml) for 24 h. The levels of IL-6 (A) and MIP-2 (B) in the medium were measured using ELISA kits. Both inhibitors significantly suppressed both the LPS stimulated release of IL-6 and MIP-2. The data are represented as mean ± SEM based on 4–6 independent experiments. * p

    Journal: PLoS ONE

    Article Title: L-Ascorbate Attenuates the Endotoxin-Induced Production of Inflammatory Mediators by Inhibiting MAPK Activation and NF-?B Translocation in Cortical Neurons/Glia Cocultures

    doi: 10.1371/journal.pone.0097276

    Figure Lengend Snippet: Effects of MAPK inhibitors on LPS-induced IL-6 and MIP-2 production. Cocultured cells were treated with 20 µM of the p38 inhibitor SB203580 or the ERK inhibitor PD98059, which were simultaneously added with LPS (100 ng/ml) for 24 h. The levels of IL-6 (A) and MIP-2 (B) in the medium were measured using ELISA kits. Both inhibitors significantly suppressed both the LPS stimulated release of IL-6 and MIP-2. The data are represented as mean ± SEM based on 4–6 independent experiments. * p

    Article Snippet: Antibodies against p38, phospho-p38 (p-p38), ERK, phospho-ERK (p-ERK), and IκB-α were purchased from Cell Signaling Technology (Beverly, MA, USA).

    Techniques: Enzyme-linked Immunosorbent Assay

    ASTX prevented steatotic primary hepatocyte apoptosis when hepatocytes subjected to HR. (A) Expressions of Bax, Bcl-2, cleaved caspase-9, caspase-9, cleaved caspase-3, caspase-3 of steatotic primary hepatocyte by hypoxia/reoxygenation injury were detected by western blot analysis. Representative blot (left) and quantified protein levels (right) are shown(n = 3). (B) Protein expression of p38 MAPK, p-p38 MAPK, ERK, p-ERK, JNK and p-JNK was detected by western blots(n = 3). α-tubulin was used as a loading control. Representative blot (up) and quantified protein levels (down) are shown. Data are expressed as the means ± SEM; *p

    Journal: PLoS ONE

    Article Title: Astaxanthin prevents ischemia-reperfusion injury of the steatotic liver in mice

    doi: 10.1371/journal.pone.0187810

    Figure Lengend Snippet: ASTX prevented steatotic primary hepatocyte apoptosis when hepatocytes subjected to HR. (A) Expressions of Bax, Bcl-2, cleaved caspase-9, caspase-9, cleaved caspase-3, caspase-3 of steatotic primary hepatocyte by hypoxia/reoxygenation injury were detected by western blot analysis. Representative blot (left) and quantified protein levels (right) are shown(n = 3). (B) Protein expression of p38 MAPK, p-p38 MAPK, ERK, p-ERK, JNK and p-JNK was detected by western blots(n = 3). α-tubulin was used as a loading control. Representative blot (up) and quantified protein levels (down) are shown. Data are expressed as the means ± SEM; *p

    Article Snippet: The primary antibodies were used are as follows: HO-1 (1:2000; Abcam, Cambridge, MA), Nrf-2 (1:2000; Santa Cruz Biotechnology), HIF-1α (1*2000; GeneTex), p-Akt (1:1000; Cell Signaling Technology), Akt (1:1000; Cell Signaling Technology), p-mTOR (1:1000; Cell Signaling Technology), mTOR (1:1000; Cell Signaling Technology), p-p38 & p38 (1:2000; Cell Signaling Technology), p-ERK1/2 & ERK1/2 (1:2000; Cell Signaling Technology), p-JNK (1:2,000; Cell Signaling Technology), JNK (1:4000; Cell Signaling Technology), α-tublin (1:4000; Santa Cruz Biotechnology) and GAPDH (1:4000; Santa Cruz Biotechnology).

    Techniques: Western Blot, Expressing

    LDIR inhibits cell growth and protein synthesis and induces bystander effects. Cell signaling pathways affected by LDIR. LDIR downregulates c-Myc and upregulates p21 WAF1/CIP1 via stimulation of TGFβ and PP2A. PP2A also inhibits mTOR signaling with repression of S6K activation and 4EBP1 phosphorylation that resulted in decrease in protein synthesis. Furthermore, LDIR induces bystander effects through p38 MAPK activation. Directly-irradiated cells release cytokine signals that affect non-irradiated (bystander) cells.

    Journal: PLoS ONE

    Article Title: Low-dose ionizing radiation exposure represses the cell cycle and protein synthesis pathways in in vitro human primary keratinocytes and U937 cell lines

    doi: 10.1371/journal.pone.0199117

    Figure Lengend Snippet: LDIR inhibits cell growth and protein synthesis and induces bystander effects. Cell signaling pathways affected by LDIR. LDIR downregulates c-Myc and upregulates p21 WAF1/CIP1 via stimulation of TGFβ and PP2A. PP2A also inhibits mTOR signaling with repression of S6K activation and 4EBP1 phosphorylation that resulted in decrease in protein synthesis. Furthermore, LDIR induces bystander effects through p38 MAPK activation. Directly-irradiated cells release cytokine signals that affect non-irradiated (bystander) cells.

    Article Snippet: The following antibodies were used: α-tubulin (Sigma-Aldrich, St Louis, MO, USA), p21WAF1/CIP1 , HIF1α (BD Biosciences, San Jose, CA, USA), c-Myc, PP2Aα, p-4EBP1, 4EBP-1, p-S6 ribosomal proteinSer235/Ser236 , S6 ribosomal protein, p-p38 MAPK, p38 MAPK, and horseradish peroxidase-linked anti-mouse and anti-rabbit IgG (all from Cell Signaling Technology).

    Techniques: Activation Assay, Irradiation

    Increased activation of stress signaling molecules in TREM2 deficient hTau mice. a-d Western blot analysis was performed on microdissected cortices ( a ) and hippocampi ( b ) of hTau ( Trem2 +/+ ) and hTau; Trem2 −/− mice using antibodies directed against phosphorylated and total JNK, ERK1/2, GSK3β, and P38 to determine relative levels of MAPK activation. Quantification of western blot data revealed highly significant increases in total levels of JNK, as well as the ratio of pJNK/total JNK, and also very highly significant increases in the ratio pGSK3β(Ser9)/GSK3β in cortex ( c ), while robust increases in total levels of GSK3β were accompanied by significant increases in the ratios pERK1/2/total ERK1/2, pGSK3β(Ser9)/Total GSK3β, pGSK3β(Y216)/total GSK3β, pP38/total P38, and pJNK/total JNK in hippocampi of 6-month mice ( d ). All experiments used n = 6 (equal males and females) mice per group unless otherwise noted. At least two independent experiments were performed for each analysis. Error bars represent SEM. ANOVA with Tukey correction *, P

    Journal: Molecular Neurodegeneration

    Article Title: TREM2 deficiency exacerbates tau pathology through dysregulated kinase signaling in a mouse model of tauopathy

    doi: 10.1186/s13024-017-0216-6

    Figure Lengend Snippet: Increased activation of stress signaling molecules in TREM2 deficient hTau mice. a-d Western blot analysis was performed on microdissected cortices ( a ) and hippocampi ( b ) of hTau ( Trem2 +/+ ) and hTau; Trem2 −/− mice using antibodies directed against phosphorylated and total JNK, ERK1/2, GSK3β, and P38 to determine relative levels of MAPK activation. Quantification of western blot data revealed highly significant increases in total levels of JNK, as well as the ratio of pJNK/total JNK, and also very highly significant increases in the ratio pGSK3β(Ser9)/GSK3β in cortex ( c ), while robust increases in total levels of GSK3β were accompanied by significant increases in the ratios pERK1/2/total ERK1/2, pGSK3β(Ser9)/Total GSK3β, pGSK3β(Y216)/total GSK3β, pP38/total P38, and pJNK/total JNK in hippocampi of 6-month mice ( d ). All experiments used n = 6 (equal males and females) mice per group unless otherwise noted. At least two independent experiments were performed for each analysis. Error bars represent SEM. ANOVA with Tukey correction *, P

    Article Snippet: After transfer, membranes were blocked with Odyssey Blocking Buffer in PBS (LI-COR Biosciences) for 1 h at room temperature and incubated in the appropriate primary antibodies in blocking buffer with added 0.1% Tween 20 overnight at 4 °C with shaking AT8 (1:5000; Pierce), AT180 (1:2000; Pierce), Tau5 (Thermo Scientific), PHF-1 (1:10,000; a generous gift from Dr. Peter Davies), MC1 (1:1000; a generous gift from Dr. Peter Davies), JNK (1:5000) and pJNK (Thr183/Tyr185; 1:1000) (Cell Signaling Technologies), ERK1/2 (1:5000) and pERK1/2 (Thr202/Tyr204; 1:2000) (Cell Signaling Technologies), GSK3β (1:5000) and pGSK3β (Ser9;1:2000) (Tyr216 1:2000) (Cell Signaling Technologies; ABCAM), p38 MAPK (1:2000) and pP38 MAPK (1:1000) (Cell Signaling Technologies), GAPDH (1:10,000; EMD Millipore) and β-Actin (1:10,000; Thermo Scientific).

    Techniques: Activation Assay, Mouse Assay, 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:

    S. pneumoniae -dependent KLF4 expression is induced by viable, replicating bacteria which undergo autolysis and are in direct contact to the host cells. Wt BMMs were stimulated with 10 6 CFU/ml viable (R6x and NaIO 4 -inactivated R6x) or 10 7 CFU/ml dead (heat- (hi) and ethanol-inactivated (eth) R6x) pneumococci ( a ). The need of bacterial replication was tested by a stimulation of wt BMMs with 10 6 CFU/ml R6x in RPMI with or without 10% FCS ( c ). Wt BMMs were stimulated with 10 6 CFU/ml R6x in direct contact or separated using a transwell system ( e ) or with R6x and R6xΔ lytA (10 6 CFU/ml each) ( g ). Cell lysates were collected 6 h after stimulation and analysed for KLF4 expression. Actin was used to confirm equal protein load. pp38 proved stimulation efficiency ( e ). All blots are representatives out of 3 independent experiments with similar results. The densitometry of the KLF4 and actin bands of the blots was quantified using Odyssey 2.0 infrared imaging system. The ratio between the KLF4 and actin densitometry was calculated. ( b ) shows the quantification of the blots of experiment ( a ), ( d ) for ( c ), ( f ) for ( e ) and ( h ) for ( g ). Data represents mean of 3 independent experiments. Differences were indicated as follows: **p

    Journal: Scientific Reports

    Article Title: DNA-release by Streptococcus pneumoniae autolysin LytA induced Krueppel-like factor 4 expression in macrophages

    doi: 10.1038/s41598-018-24152-1

    Figure Lengend Snippet: S. pneumoniae -dependent KLF4 expression is induced by viable, replicating bacteria which undergo autolysis and are in direct contact to the host cells. Wt BMMs were stimulated with 10 6 CFU/ml viable (R6x and NaIO 4 -inactivated R6x) or 10 7 CFU/ml dead (heat- (hi) and ethanol-inactivated (eth) R6x) pneumococci ( a ). The need of bacterial replication was tested by a stimulation of wt BMMs with 10 6 CFU/ml R6x in RPMI with or without 10% FCS ( c ). Wt BMMs were stimulated with 10 6 CFU/ml R6x in direct contact or separated using a transwell system ( e ) or with R6x and R6xΔ lytA (10 6 CFU/ml each) ( g ). Cell lysates were collected 6 h after stimulation and analysed for KLF4 expression. Actin was used to confirm equal protein load. pp38 proved stimulation efficiency ( e ). All blots are representatives out of 3 independent experiments with similar results. The densitometry of the KLF4 and actin bands of the blots was quantified using Odyssey 2.0 infrared imaging system. The ratio between the KLF4 and actin densitometry was calculated. ( b ) shows the quantification of the blots of experiment ( a ), ( d ) for ( c ), ( f ) for ( e ) and ( h ) for ( g ). Data represents mean of 3 independent experiments. Differences were indicated as follows: **p

    Article Snippet: Membranes were exposed to antibodies specific for mouse KLF4 (GKLF antibody (H-180) rabbit polyclonal IgG, sc-20691), actin, pp38 (all Santa Cruz Biotechnology, Inc., Heidelberg, Germany) overnight at 4 °C and subsequently incubated with secondary antibodies (anti-goat or anti-rabbit either Cy5.5- or IRDye 800–labeled (Rockland, Limerick, PA, USA)) for 1 h at room temperature.

    Techniques: Expressing, Imaging

    lncRNA-ROR mediated myocardial hypoxia/reoxygenation (H/R) by regulating the p38/MAPK pathway. H9c2 cells and human cardiomyocytes (HCM) were transfected with lncRNA-ROR overexpression vector (lncRNA-ROR) and inhibition vector (si-lncRNA-ROR). After H/R treatment for 24 h, ( A ) the protein levels of ERK and p38 were examined by western blot and ( B ) the mRNA expressions of ERK and p38 were determined by qRT-PCR. Data are reported as means±SE. *P

    Journal: Brazilian Journal of Medical and Biological Research

    Article Title: Long non-coding RNA-ROR aggravates myocardial ischemia/reperfusion injury

    doi: 10.1590/1414-431X20186555

    Figure Lengend Snippet: lncRNA-ROR mediated myocardial hypoxia/reoxygenation (H/R) by regulating the p38/MAPK pathway. H9c2 cells and human cardiomyocytes (HCM) were transfected with lncRNA-ROR overexpression vector (lncRNA-ROR) and inhibition vector (si-lncRNA-ROR). After H/R treatment for 24 h, ( A ) the protein levels of ERK and p38 were examined by western blot and ( B ) the mRNA expressions of ERK and p38 were determined by qRT-PCR. Data are reported as means±SE. *P

    Article Snippet: After blocking with 8% milk in PBS, pH 7.5, the membranes were incubated with the following specific primary antibodies of Bax (ab32503), Bcl-2 (ab59348), cytochrome C (ab13575), Smac/Diablo (ab32023), cleaved-capase-3 (ab13847), cleaved-capase-9 (ab2324), p-p38 (ab47363), p38 (ab31828), p-ERK (ab214362), and ERK1/2 (ab196883; all at a dilution of 1:1000, Abcam, UK).

    Techniques: Transfection, Over Expression, Plasmid Preparation, Inhibition, Western Blot, Quantitative RT-PCR

    SGE regulates AKT and p38 MAPK phosphorylation at the beginning of myogenesis. C2C12 cells were cultured in GM. To begin the differentiation, this medium was withdrawn and then replaced by DM. Treatment was started by adding 10 nM of SGE or 1 nM of synthetic 1α,25(|OH) 2 D 3 (1,25D) in DM during different periods of time (1–48 h). Western blots were performed with anti p-AKT, total AKT, p-p38 MAPK, and p38α and tubulin antibodies. Tubulin was used as loading protein marker. Representative blots from three independent experiments (upper panel) and their quantification (lower panel) are shown. Protein bands quantification was done using ImageJ. Results were then represented in bar graphs as phosphorylated protein normalized with its corresponding total from treated conditions and referred to control (Fold) (lower panel). Significant differences between conditions at each time point were analyzed by Student’s t -test, ** P

    Journal: Biology Open

    Article Title: 1α,25(OH)2D3-glycosides from Solanum glaucophyllum leaves extract induce myoblasts differentiation through p38 MAPK and AKT activation

    doi: 10.1242/bio.033670

    Figure Lengend Snippet: SGE regulates AKT and p38 MAPK phosphorylation at the beginning of myogenesis. C2C12 cells were cultured in GM. To begin the differentiation, this medium was withdrawn and then replaced by DM. Treatment was started by adding 10 nM of SGE or 1 nM of synthetic 1α,25(|OH) 2 D 3 (1,25D) in DM during different periods of time (1–48 h). Western blots were performed with anti p-AKT, total AKT, p-p38 MAPK, and p38α and tubulin antibodies. Tubulin was used as loading protein marker. Representative blots from three independent experiments (upper panel) and their quantification (lower panel) are shown. Protein bands quantification was done using ImageJ. Results were then represented in bar graphs as phosphorylated protein normalized with its corresponding total from treated conditions and referred to control (Fold) (lower panel). Significant differences between conditions at each time point were analyzed by Student’s t -test, ** P

    Article Snippet: The antibodies used were: anti-p-p38 MAPK (4511), p-AKT (9271), AKT (4685) from Cell Signaling Technology; Goat anti-Rabbit, IgG-HRP (sc-2004) and anti-β-actin (sc-47778) antibodies from Santa Cruz Biotechnology.

    Techniques: Cell Culture, Western Blot, Marker

    Cell cycle progression of C2C12 skeletal muscle cells after SGE treatment: role of p38 MAPK. A. This panel shows a representative histogram and DNA quantification from three independent experiments showing the percentage of cells in G0/G1-, S- and G2/M-phases (Y-axis label) ±s.d. Data were analyzed by one way ANOVA, followed by t -test, ** P

    Journal: Biology Open

    Article Title: 1α,25(OH)2D3-glycosides from Solanum glaucophyllum leaves extract induce myoblasts differentiation through p38 MAPK and AKT activation

    doi: 10.1242/bio.033670

    Figure Lengend Snippet: Cell cycle progression of C2C12 skeletal muscle cells after SGE treatment: role of p38 MAPK. A. This panel shows a representative histogram and DNA quantification from three independent experiments showing the percentage of cells in G0/G1-, S- and G2/M-phases (Y-axis label) ±s.d. Data were analyzed by one way ANOVA, followed by t -test, ** P

    Article Snippet: The antibodies used were: anti-p-p38 MAPK (4511), p-AKT (9271), AKT (4685) from Cell Signaling Technology; Goat anti-Rabbit, IgG-HRP (sc-2004) and anti-β-actin (sc-47778) antibodies from Santa Cruz Biotechnology.

    Techniques:

    (IVIg + LPS)-induced MAPK phosphorylation is lower in monocytes from people with the FcγRIIA risk variant. (A) Monocytes from healthy participants with the non-risk genotype (CC) or the risk genotype (TT) were unstimulated or stimulated with LPS (100 ng/ml), IVIg (5 mg/ml), or both, for 0, 10, 40, or 120 min. Cell lysates (2.5 × 10 5 cells / time point) were prepared at the indicated times. Lysates were separated by SDS-PAGE and analyzed by western blotting using phosphospecific antibodies for ERK1/2, p38, and using GAPDH, as a loading control. Representative western blot from participants with the TT genotype are shown in (A) . Results are representative of n = 3 experiments per genotype; monocytes were derived from 1 participant for each of 3 independent experiments. Densitometry for pERK1/2 and pp38; normalized to GAPDH and relative to LPS 10 min; are averaged from n = 3 independent experiments and shown below each band and values are graphed as mean ± SEM for each genotype. Monocytes were pre-treated for 1 h with an appropriate volume of DMSO, as a vehicle control, or (B) the ERK1/2 inhibitors, PD98059 (PD) and SCH772984 (SCH); or (C) the p38 inhibitors, SB203580 (SB) and BIRB796 (BIR) and were unstimulated (C) or stimulated with LPS (100 ng/ml), IVIg (5 mg/ml), or both for 24 h. Clarified cell supernatants were analyzed for IL-10 by ELISA. (B,C) Values are reported as mean ± SEM for n = 7 participants for the CC genotype and n = 4 participants for the TT genotype performed as independent experiments, and assayed in duplicate. * p

    Journal: Frontiers in Immunology

    Article Title: IVIg and LPS Co-stimulation Induces IL-10 Production by Human Monocytes, Which Is Compromised by an FcγRIIA Disease-Associated Gene Variant

    doi: 10.3389/fimmu.2018.02676

    Figure Lengend Snippet: (IVIg + LPS)-induced MAPK phosphorylation is lower in monocytes from people with the FcγRIIA risk variant. (A) Monocytes from healthy participants with the non-risk genotype (CC) or the risk genotype (TT) were unstimulated or stimulated with LPS (100 ng/ml), IVIg (5 mg/ml), or both, for 0, 10, 40, or 120 min. Cell lysates (2.5 × 10 5 cells / time point) were prepared at the indicated times. Lysates were separated by SDS-PAGE and analyzed by western blotting using phosphospecific antibodies for ERK1/2, p38, and using GAPDH, as a loading control. Representative western blot from participants with the TT genotype are shown in (A) . Results are representative of n = 3 experiments per genotype; monocytes were derived from 1 participant for each of 3 independent experiments. Densitometry for pERK1/2 and pp38; normalized to GAPDH and relative to LPS 10 min; are averaged from n = 3 independent experiments and shown below each band and values are graphed as mean ± SEM for each genotype. Monocytes were pre-treated for 1 h with an appropriate volume of DMSO, as a vehicle control, or (B) the ERK1/2 inhibitors, PD98059 (PD) and SCH772984 (SCH); or (C) the p38 inhibitors, SB203580 (SB) and BIRB796 (BIR) and were unstimulated (C) or stimulated with LPS (100 ng/ml), IVIg (5 mg/ml), or both for 24 h. Clarified cell supernatants were analyzed for IL-10 by ELISA. (B,C) Values are reported as mean ± SEM for n = 7 participants for the CC genotype and n = 4 participants for the TT genotype performed as independent experiments, and assayed in duplicate. * p

    Article Snippet: Antibodies used for western blot analyses for MAPK activation experiments were anti-pERK1/2 (Cell Signaling Technology, 9,106), anti-pp38 (Cell Signaling Technology, 4,631), and anti-GAPDH (Fitzgerald Industries International, 10R-G109a, Acton, MA, USA).

    Techniques: Variant Assay, SDS Page, Western Blot, Derivative Assay, Enzyme-linked Immunosorbent Assay

    MAPKs are required for IVIg-induced IL-10 production in response to LPS. (A) Monocytes from healthy control participants were unstimulated or stimulated with LPS (100 ng/ml), IVIg (5 mg/ml), or both, for 0, 10, 40, or 120 min. Cell lysates (2.5 × 10 5 cells / time point) were prepared at the indicated times. Lysates were separated by SDS-PAGE and analyzed by western blotting using phosphospecific antibodies for ERK1/2 and p38, and GAPDH, as a loading control. Results are representative of n = 5 experiments; monocytes were derived from 1 participant for each of 5 independent experiments. Densitometry for pERK1/2 and pp38; normalized to GAPDH and relative to LPS 10 min; are averaged and shown below each band and values are graphed and reported as mean ± SEM. In (B) and (C) , monocytes were pre-treated for 1 h with an appropriate volume of DMSO, as a vehicle control, or (B) the ERK1/2 inhibitors, PD98059 (PD) and SCH772984 (SCH), or (C) the p38 inhibitors, SB203580 (SB), and BIRB796 (BIR). In (D) and (E) monocytes were untreated (UnRx) or pre-treated for 48 h with a non-silencing siRNA (ns) or 2 different sets of siRNAs (si1 or si2) to ERK1 and 2 (D) or p38α, p38γ, and p38δ (E) . Samples in (D) and (E) were prepared, as above, and analyzed by western blotting using antibodies for ERK1/2 (D) or p38 (E) and GAPDH, as a loading control. Densitometry for ERK1/2 (D) or p38 (E) normalized to GAPDH and relative to untreated control (UnRx) are shown below each band. Monocytes in (B–E) were unstimulated (C) or stimulated with LPS (100 ng/ml), IVIg (5 mg/ml), or (IVIg + LPS) for 24 h. Clarified cell supernatants were assayed for IL-10 by ELISA. Values are reported as mean ± SEM for n = 7 (B,C) or n = 5 or 6 (D,E) participants performed as independent experiments, assayed in duplicate. * p

    Journal: Frontiers in Immunology

    Article Title: IVIg and LPS Co-stimulation Induces IL-10 Production by Human Monocytes, Which Is Compromised by an FcγRIIA Disease-Associated Gene Variant

    doi: 10.3389/fimmu.2018.02676

    Figure Lengend Snippet: MAPKs are required for IVIg-induced IL-10 production in response to LPS. (A) Monocytes from healthy control participants were unstimulated or stimulated with LPS (100 ng/ml), IVIg (5 mg/ml), or both, for 0, 10, 40, or 120 min. Cell lysates (2.5 × 10 5 cells / time point) were prepared at the indicated times. Lysates were separated by SDS-PAGE and analyzed by western blotting using phosphospecific antibodies for ERK1/2 and p38, and GAPDH, as a loading control. Results are representative of n = 5 experiments; monocytes were derived from 1 participant for each of 5 independent experiments. Densitometry for pERK1/2 and pp38; normalized to GAPDH and relative to LPS 10 min; are averaged and shown below each band and values are graphed and reported as mean ± SEM. In (B) and (C) , monocytes were pre-treated for 1 h with an appropriate volume of DMSO, as a vehicle control, or (B) the ERK1/2 inhibitors, PD98059 (PD) and SCH772984 (SCH), or (C) the p38 inhibitors, SB203580 (SB), and BIRB796 (BIR). In (D) and (E) monocytes were untreated (UnRx) or pre-treated for 48 h with a non-silencing siRNA (ns) or 2 different sets of siRNAs (si1 or si2) to ERK1 and 2 (D) or p38α, p38γ, and p38δ (E) . Samples in (D) and (E) were prepared, as above, and analyzed by western blotting using antibodies for ERK1/2 (D) or p38 (E) and GAPDH, as a loading control. Densitometry for ERK1/2 (D) or p38 (E) normalized to GAPDH and relative to untreated control (UnRx) are shown below each band. Monocytes in (B–E) were unstimulated (C) or stimulated with LPS (100 ng/ml), IVIg (5 mg/ml), or (IVIg + LPS) for 24 h. Clarified cell supernatants were assayed for IL-10 by ELISA. Values are reported as mean ± SEM for n = 7 (B,C) or n = 5 or 6 (D,E) participants performed as independent experiments, assayed in duplicate. * p

    Article Snippet: Antibodies used for western blot analyses for MAPK activation experiments were anti-pERK1/2 (Cell Signaling Technology, 9,106), anti-pp38 (Cell Signaling Technology, 4,631), and anti-GAPDH (Fitzgerald Industries International, 10R-G109a, Acton, MA, USA).

    Techniques: SDS Page, Western Blot, Derivative Assay, Enzyme-linked Immunosorbent Assay

    Phosphorylation of the MAPKs p38, ERK1/2 and JNK is increased upon IV/ S. aureus super-infection. Calu-3 ( a – d ) or A549 ( e – i ) cells were infected with IV H1N1(M) ( a – h ) or H3N2 ( i ) (MOI 5) for 0.5 h and super-infected with S. aureus 6850 ( a – i ), SH1000 or MRSA USA300 ( i ) (MOI 50). Extracellular bacteria were removed by gentamicin treatment 3 h after bacterial infection. At 8 h p.i. cells were lysed and whole cell lysates were subjected to Western blot analysis monitoring P-p38, P-ERK1/2, P-JNK, PGN and PB1. Equal protein loading was verified by detection of p38, ERK2 and JNK ( a , e , i ). Original blots are shown in Supplementary Fig. S8b (upper panel: Fig. 5a, lower panel: Fig. 5e) and S8c (upper panel: Fig. 5i [S. aureus 6850], lower panel [ S. aureus SH1000, USA300]). Relative levels of P-p38, P-ERK1/2 and P-JNK compared to p38, ERK2 or JNK bands, respectively, were quantified by using ImageJ 2006.02.01 software ( b – d , f – h ). Means ± SD of at least three independent experiments are shown. Two-tailed unpaired t-tests were performed for comparison of IV H1N1(M)-infected and IV H1N1(M)/ S. aureus 6850 super-infected samples (*p

    Journal: Scientific Reports

    Article Title: Mitogen-activated protein kinases (MAPKs) regulate IL-6 over-production during concomitant influenza virus and Staphylococcus aureus infection

    doi: 10.1038/srep42473

    Figure Lengend Snippet: Phosphorylation of the MAPKs p38, ERK1/2 and JNK is increased upon IV/ S. aureus super-infection. Calu-3 ( a – d ) or A549 ( e – i ) cells were infected with IV H1N1(M) ( a – h ) or H3N2 ( i ) (MOI 5) for 0.5 h and super-infected with S. aureus 6850 ( a – i ), SH1000 or MRSA USA300 ( i ) (MOI 50). Extracellular bacteria were removed by gentamicin treatment 3 h after bacterial infection. At 8 h p.i. cells were lysed and whole cell lysates were subjected to Western blot analysis monitoring P-p38, P-ERK1/2, P-JNK, PGN and PB1. Equal protein loading was verified by detection of p38, ERK2 and JNK ( a , e , i ). Original blots are shown in Supplementary Fig. S8b (upper panel: Fig. 5a, lower panel: Fig. 5e) and S8c (upper panel: Fig. 5i [S. aureus 6850], lower panel [ S. aureus SH1000, USA300]). Relative levels of P-p38, P-ERK1/2 and P-JNK compared to p38, ERK2 or JNK bands, respectively, were quantified by using ImageJ 2006.02.01 software ( b – d , f – h ). Means ± SD of at least three independent experiments are shown. Two-tailed unpaired t-tests were performed for comparison of IV H1N1(M)-infected and IV H1N1(M)/ S. aureus 6850 super-infected samples (*p

    Article Snippet: Cellular proteins were monitored by usage of P-p38 (T180/Y182, 1:500), P-JNK (T183/Y185, 1:500) (BD Transduction Technology, San Jose, USA), P-ERK1/2 (T202/Y204, 1:1000), JNK (1:500), ERK1/2 (1:1000), MyD88 (1:1000) (Cell Signalling Technology), p38 (C-20, 1:500), ERK2 (C-14, 1:1000) (Santa Cruz Biotechnology, Dallas, USA) and α-Tubulin (DM1A, 1:1000) (Sigma) antibodies.

    Techniques: Infection, Western Blot, Software, Two Tailed Test

    Specific inhibition of the MAPK p38 or the Raf/MEK/ERK signalling pathway reduces IL - 6 mRNA levels during super-infection. A549 cells were treated with 10 μM p38 inhibitor (SB202190) ( a ), 10 μM MEK inhibitor (U0126) ( b ), 10 μM JNK inhibitor (SP600125) ( c ) or DMSO as solvent control for 0.5 h. A549 cells were transfected with 33 nM siRNA directed against p38 ( d ), ERK1/2 ( e ) or control siRNA and incubated for 48 h. Subsequently, cells were infected with IV H1N1(M) (MOI 5) for 0.5 h and super-infected with S. aureus 6850 (MOI 50) ( a – e ), in the presence of the inhibitors ( a – c ). Extracellular bacteria were removed by gentamicin treatment 3 h after bacterial infection ( a – e ), and supplemented with the specific inhibitors ( a – c ). Expression of p38 ( d ) and ERK1/2 ( e ) was monitored by Western blot analysis (original blots are depicted in Supplementary Fig. S8d ). IL - 6 mRNA levels were measured in duplicates at 8 h p.i. Means ± SD of at least three independent experiments are shown. IV-infected samples of control cells were arbitrarily set as 100%. After normalisation, one-way ANOVA followed by Dunnett’s multiple comparison tests were performed for comparison of IV H1N1(M)-infected and IV H1N1(M)/ S. aureus 6850 super-infected samples, control and treated IV-infected samples, and control and treated super-infected samples (*p

    Journal: Scientific Reports

    Article Title: Mitogen-activated protein kinases (MAPKs) regulate IL-6 over-production during concomitant influenza virus and Staphylococcus aureus infection

    doi: 10.1038/srep42473

    Figure Lengend Snippet: Specific inhibition of the MAPK p38 or the Raf/MEK/ERK signalling pathway reduces IL - 6 mRNA levels during super-infection. A549 cells were treated with 10 μM p38 inhibitor (SB202190) ( a ), 10 μM MEK inhibitor (U0126) ( b ), 10 μM JNK inhibitor (SP600125) ( c ) or DMSO as solvent control for 0.5 h. A549 cells were transfected with 33 nM siRNA directed against p38 ( d ), ERK1/2 ( e ) or control siRNA and incubated for 48 h. Subsequently, cells were infected with IV H1N1(M) (MOI 5) for 0.5 h and super-infected with S. aureus 6850 (MOI 50) ( a – e ), in the presence of the inhibitors ( a – c ). Extracellular bacteria were removed by gentamicin treatment 3 h after bacterial infection ( a – e ), and supplemented with the specific inhibitors ( a – c ). Expression of p38 ( d ) and ERK1/2 ( e ) was monitored by Western blot analysis (original blots are depicted in Supplementary Fig. S8d ). IL - 6 mRNA levels were measured in duplicates at 8 h p.i. Means ± SD of at least three independent experiments are shown. IV-infected samples of control cells were arbitrarily set as 100%. After normalisation, one-way ANOVA followed by Dunnett’s multiple comparison tests were performed for comparison of IV H1N1(M)-infected and IV H1N1(M)/ S. aureus 6850 super-infected samples, control and treated IV-infected samples, and control and treated super-infected samples (*p

    Article Snippet: Cellular proteins were monitored by usage of P-p38 (T180/Y182, 1:500), P-JNK (T183/Y185, 1:500) (BD Transduction Technology, San Jose, USA), P-ERK1/2 (T202/Y204, 1:1000), JNK (1:500), ERK1/2 (1:1000), MyD88 (1:1000) (Cell Signalling Technology), p38 (C-20, 1:500), ERK2 (C-14, 1:1000) (Santa Cruz Biotechnology, Dallas, USA) and α-Tubulin (DM1A, 1:1000) (Sigma) antibodies.

    Techniques: Inhibition, Infection, Transfection, Incubation, Expressing, Western Blot

    Effect of TPA on fisetin-induced inhibition of uPA expression, cell migration and invasion in SiHa cells. Cells were pretreated with or without fisetin for 2 h, and then treated with or without 50 ng/ml of TPA for 24 h. (A) The phosphorylated protein and total protein of p38 MAPK were determined by Western blotting. (B) The mRNA level of uPA after each treatment was examined by RT-PCR. (C) The protein level of uPA was determined by Western blotting. β-actin was used as the internal control. (D) The conditioned medium from each treatment was collected, and the uPA activity was determined by casein zymography. The migratory (E) and invasive (F) ability of SiHa cells after treatment were determined. Migrating and invading cells were photographed using phase-contrast microscopy. Values represent mean ± SD of three independent experiments. *, P

    Journal: PLoS ONE

    Article Title: Fisetin Inhibits Migration and Invasion of Human Cervical Cancer Cells by Down-Regulating Urokinase Plasminogen Activator Expression through Suppressing the p38 MAPK-Dependent NF-?B Signaling Pathway

    doi: 10.1371/journal.pone.0071983

    Figure Lengend Snippet: Effect of TPA on fisetin-induced inhibition of uPA expression, cell migration and invasion in SiHa cells. Cells were pretreated with or without fisetin for 2 h, and then treated with or without 50 ng/ml of TPA for 24 h. (A) The phosphorylated protein and total protein of p38 MAPK were determined by Western blotting. (B) The mRNA level of uPA after each treatment was examined by RT-PCR. (C) The protein level of uPA was determined by Western blotting. β-actin was used as the internal control. (D) The conditioned medium from each treatment was collected, and the uPA activity was determined by casein zymography. The migratory (E) and invasive (F) ability of SiHa cells after treatment were determined. Migrating and invading cells were photographed using phase-contrast microscopy. Values represent mean ± SD of three independent experiments. *, P

    Article Snippet: The antibodies against p-ERK1/2, ERK1/2, p-p38 MAPK, p38 MAPK, p-JNK, JNK1/2, uPA, α-tubulin, NF-κB (p65) and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

    Techniques: Inhibition, Expressing, Migration, Western Blot, Reverse Transcription Polymerase Chain Reaction, Activity Assay, Zymography, Microscopy

    The proposed fisetin model in inhibiting the migration and invasion of cervical cancer cells. Fisetin inhibits the phosphorylation of p38 MAPK and impairs translocation of NF-κB to the nucleus. The decreased NF-κB in the nucleus reduces its binding on the promoter of the uPA gene, and results in repressing the expression and activity of uPA, thereby disrupting the migratory and invasive ability of cervical cancer SiHa cells.

    Journal: PLoS ONE

    Article Title: Fisetin Inhibits Migration and Invasion of Human Cervical Cancer Cells by Down-Regulating Urokinase Plasminogen Activator Expression through Suppressing the p38 MAPK-Dependent NF-?B Signaling Pathway

    doi: 10.1371/journal.pone.0071983

    Figure Lengend Snippet: The proposed fisetin model in inhibiting the migration and invasion of cervical cancer cells. Fisetin inhibits the phosphorylation of p38 MAPK and impairs translocation of NF-κB to the nucleus. The decreased NF-κB in the nucleus reduces its binding on the promoter of the uPA gene, and results in repressing the expression and activity of uPA, thereby disrupting the migratory and invasive ability of cervical cancer SiHa cells.

    Article Snippet: The antibodies against p-ERK1/2, ERK1/2, p-p38 MAPK, p38 MAPK, p-JNK, JNK1/2, uPA, α-tubulin, NF-κB (p65) and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

    Techniques: Migration, Translocation Assay, Binding Assay, Expressing, Activity Assay

    Effects of the inhibitor of p38 MAPK on fisetin-induced inhibition of uPA expression, cell migration and invasion in SiHa cells. Cells were pretreated with or without 25 µM of a p38 inhibitor, SB203580 for 2 h, and then treated with or without 20 µM of fisetin, as indicated, for another 48 h. (A) The mRNA level of uPA after each treatment was examined by RT-PCR. (B) The protein level of uPA was determined by Western blotting. β-actin was used as the internal control. (C) Cells were fixed, permeabilized, and immuno-stained with anti-uPA antibody (red) and cell nuclei were counter-stained with DAPI reagent (blue). (D) The conditioned medium from each treatment was collected, and the uPA activity was determined by casein zymography. The migratory (E) and invasive (F) ability of SiHa cells after treatment were determined. Migrating and invading cells were photographed using phase-contrast microscopy. Bars show the value as mean ± S.E. from three independent experiments. *, P

    Journal: PLoS ONE

    Article Title: Fisetin Inhibits Migration and Invasion of Human Cervical Cancer Cells by Down-Regulating Urokinase Plasminogen Activator Expression through Suppressing the p38 MAPK-Dependent NF-?B Signaling Pathway

    doi: 10.1371/journal.pone.0071983

    Figure Lengend Snippet: Effects of the inhibitor of p38 MAPK on fisetin-induced inhibition of uPA expression, cell migration and invasion in SiHa cells. Cells were pretreated with or without 25 µM of a p38 inhibitor, SB203580 for 2 h, and then treated with or without 20 µM of fisetin, as indicated, for another 48 h. (A) The mRNA level of uPA after each treatment was examined by RT-PCR. (B) The protein level of uPA was determined by Western blotting. β-actin was used as the internal control. (C) Cells were fixed, permeabilized, and immuno-stained with anti-uPA antibody (red) and cell nuclei were counter-stained with DAPI reagent (blue). (D) The conditioned medium from each treatment was collected, and the uPA activity was determined by casein zymography. The migratory (E) and invasive (F) ability of SiHa cells after treatment were determined. Migrating and invading cells were photographed using phase-contrast microscopy. Bars show the value as mean ± S.E. from three independent experiments. *, P

    Article Snippet: The antibodies against p-ERK1/2, ERK1/2, p-p38 MAPK, p38 MAPK, p-JNK, JNK1/2, uPA, α-tubulin, NF-κB (p65) and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

    Techniques: Inhibition, Expressing, Migration, Reverse Transcription Polymerase Chain Reaction, Western Blot, Staining, Activity Assay, Zymography, Microscopy

    Schematic representation of proposed signaling pathways suggested by the results of this study. Methylglyoxal increases production of reactive oxygen species (ROS), which then reduces the mitochondrial membrane potential (MMP) and ATP production via UCP2 upregulation. UCP2 upregulation in turn leads to β -cell damage and, ultimately, impairment of insulin secretion. ROS also may induce β -cell damage and impair insulin secretion directly via upregulation of MAPKs (JNK/P38).

    Journal: Journal of Diabetes Research

    Article Title: Methylglyoxal Impairs Insulin Secretion of Pancreatic β-Cells through Increased Production of ROS and Mitochondrial Dysfunction Mediated by Upregulation of UCP2 and MAPKs

    doi: 10.1155/2016/2029854

    Figure Lengend Snippet: Schematic representation of proposed signaling pathways suggested by the results of this study. Methylglyoxal increases production of reactive oxygen species (ROS), which then reduces the mitochondrial membrane potential (MMP) and ATP production via UCP2 upregulation. UCP2 upregulation in turn leads to β -cell damage and, ultimately, impairment of insulin secretion. ROS also may induce β -cell damage and impair insulin secretion directly via upregulation of MAPKs (JNK/P38).

    Article Snippet: Determination of UCP2, p-JNK, JNK, p-P38, and P38 Protein Expression After incubation with various concentrations of MG (0.05 mM or 0.1 mM) or H2 O2 (0.2 mM) with or without NAC (0.6 mM) for 1, 2, and 3 hours, MIN6 cells were washed with PBS and subjected to lysis in radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Institute of Biotechnology).

    Techniques:

    Time-dependent effects of MG on expression of p-JNK, JNK, p-P38, and P38 proteins in MIN6 cells. The expression of p-JNK and JNK (a) and p-P38 and P38 (d) was increased after treatment with MG for 1 h. Cells were incubated with 0.1 mM MG for 1, 2, and 3 h. Results shown in (b), (c), (e), and (f) are fold change compared to baseline (0 mM MG). ∗ p

    Journal: Journal of Diabetes Research

    Article Title: Methylglyoxal Impairs Insulin Secretion of Pancreatic β-Cells through Increased Production of ROS and Mitochondrial Dysfunction Mediated by Upregulation of UCP2 and MAPKs

    doi: 10.1155/2016/2029854

    Figure Lengend Snippet: Time-dependent effects of MG on expression of p-JNK, JNK, p-P38, and P38 proteins in MIN6 cells. The expression of p-JNK and JNK (a) and p-P38 and P38 (d) was increased after treatment with MG for 1 h. Cells were incubated with 0.1 mM MG for 1, 2, and 3 h. Results shown in (b), (c), (e), and (f) are fold change compared to baseline (0 mM MG). ∗ p

    Article Snippet: Determination of UCP2, p-JNK, JNK, p-P38, and P38 Protein Expression After incubation with various concentrations of MG (0.05 mM or 0.1 mM) or H2 O2 (0.2 mM) with or without NAC (0.6 mM) for 1, 2, and 3 hours, MIN6 cells were washed with PBS and subjected to lysis in radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Institute of Biotechnology).

    Techniques: Expressing, Incubation

    Effects of MG on expression of p-JNK, JNK, p-P38, and P38 proteins in MIN6 cells. The expression of p-JNK and JNK (a) and p-P38 and P38 (d) was increased by MG. Cells were incubated with 0.05 or 0.1 mM MG for 3 h. Results shown in (b), (c), (e), and (f) are fold change compared to baseline (0 mM MG + 0 mM NAC). The effects of MG on MIN6 were reversed by coincubation with NAC. ∗ p

    Journal: Journal of Diabetes Research

    Article Title: Methylglyoxal Impairs Insulin Secretion of Pancreatic β-Cells through Increased Production of ROS and Mitochondrial Dysfunction Mediated by Upregulation of UCP2 and MAPKs

    doi: 10.1155/2016/2029854

    Figure Lengend Snippet: Effects of MG on expression of p-JNK, JNK, p-P38, and P38 proteins in MIN6 cells. The expression of p-JNK and JNK (a) and p-P38 and P38 (d) was increased by MG. Cells were incubated with 0.05 or 0.1 mM MG for 3 h. Results shown in (b), (c), (e), and (f) are fold change compared to baseline (0 mM MG + 0 mM NAC). The effects of MG on MIN6 were reversed by coincubation with NAC. ∗ p

    Article Snippet: Determination of UCP2, p-JNK, JNK, p-P38, and P38 Protein Expression After incubation with various concentrations of MG (0.05 mM or 0.1 mM) or H2 O2 (0.2 mM) with or without NAC (0.6 mM) for 1, 2, and 3 hours, MIN6 cells were washed with PBS and subjected to lysis in radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Institute of Biotechnology).

    Techniques: Expressing, Incubation