p38  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc p38
    Effects of pilloin on MAPK signalling pathways in LPS-stimulated RAW 264.7 macrophages. RAW 264.7 cells (10 6 cells in MP-6 plates) were treated with vehicle, pilloin or the indicated inhibitor for 6 h before incubation with LPS (100 ng/mL) for 20 min. Cell lysates were analysed by Western blot to detect the activation of ( A ) JNK, ( B ) ERK and ( C ) <t>p38.</t> PD98059 (ERK inhibitor), SB203580 (p38 inhibitor) and SP600125 (JNK inhibitor) served as positive controls. * indicates a significant difference versus the LPS-treated group ( p
    P38, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Pilloin, A Flavonoid Isolated from Aquilaria sinensis, Exhibits Anti-Inflammatory Activity In Vitro and In Vivo"

    Article Title: Pilloin, A Flavonoid Isolated from Aquilaria sinensis, Exhibits Anti-Inflammatory Activity In Vitro and In Vivo

    Journal: Molecules

    doi: 10.3390/molecules23123177

    Effects of pilloin on MAPK signalling pathways in LPS-stimulated RAW 264.7 macrophages. RAW 264.7 cells (10 6 cells in MP-6 plates) were treated with vehicle, pilloin or the indicated inhibitor for 6 h before incubation with LPS (100 ng/mL) for 20 min. Cell lysates were analysed by Western blot to detect the activation of ( A ) JNK, ( B ) ERK and ( C ) p38. PD98059 (ERK inhibitor), SB203580 (p38 inhibitor) and SP600125 (JNK inhibitor) served as positive controls. * indicates a significant difference versus the LPS-treated group ( p
    Figure Legend Snippet: Effects of pilloin on MAPK signalling pathways in LPS-stimulated RAW 264.7 macrophages. RAW 264.7 cells (10 6 cells in MP-6 plates) were treated with vehicle, pilloin or the indicated inhibitor for 6 h before incubation with LPS (100 ng/mL) for 20 min. Cell lysates were analysed by Western blot to detect the activation of ( A ) JNK, ( B ) ERK and ( C ) p38. PD98059 (ERK inhibitor), SB203580 (p38 inhibitor) and SP600125 (JNK inhibitor) served as positive controls. * indicates a significant difference versus the LPS-treated group ( p

    Techniques Used: Incubation, Western Blot, Activation Assay

    2) Product Images from "Isosteviol Derivative Inhibits Osteoclast Differentiation and Ameliorates Ovariectomy-Induced Osteoporosis"

    Article Title: Isosteviol Derivative Inhibits Osteoclast Differentiation and Ameliorates Ovariectomy-Induced Osteoporosis

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-29257-1

    The inhibitory effects of NC-8 on the RANKL-induced osteoclast differentiation signaling pathway. ( A and B ) RAW 264.7 cells were treated with 100 ng/ml RANKL or with 10 μg/ml NC-8, and the protein was collected after the reaction. Western blot was used to detect MAPK pathway and downstream NFATc1, c-Fos and NF-κB protein molecules. ( A ) Relative protein expressions analysis of proteins related to the MAPK signaling pathway, including p-ERK, p-JNK, and p-p38. β-actin was used as a loading control. ( B ) Relative protein expressions analysis of proteins related to the NF-κB signaling pathway, including p65, NFATc1 and c-Fos. β-actin was used as a loading control. Relative expression levels of p65, NFATc1 and c-Fos protein in the cytoplasm and in the nuclear fractions. β-actin and HDAC1 were used as loading controls. The results are expressed as the mean ± S.E.M. of four independent experiments. *p
    Figure Legend Snippet: The inhibitory effects of NC-8 on the RANKL-induced osteoclast differentiation signaling pathway. ( A and B ) RAW 264.7 cells were treated with 100 ng/ml RANKL or with 10 μg/ml NC-8, and the protein was collected after the reaction. Western blot was used to detect MAPK pathway and downstream NFATc1, c-Fos and NF-κB protein molecules. ( A ) Relative protein expressions analysis of proteins related to the MAPK signaling pathway, including p-ERK, p-JNK, and p-p38. β-actin was used as a loading control. ( B ) Relative protein expressions analysis of proteins related to the NF-κB signaling pathway, including p65, NFATc1 and c-Fos. β-actin was used as a loading control. Relative expression levels of p65, NFATc1 and c-Fos protein in the cytoplasm and in the nuclear fractions. β-actin and HDAC1 were used as loading controls. The results are expressed as the mean ± S.E.M. of four independent experiments. *p

    Techniques Used: Western Blot, Expressing

    3) Product Images from "Follistatin-Like 1 Promotes Bleomycin-Induced Pulmonary Fibrosis through the Transforming Growth Factor Beta 1/Mitogen-Activated Protein Kinase Signaling Pathway"

    Article Title: Follistatin-Like 1 Promotes Bleomycin-Induced Pulmonary Fibrosis through the Transforming Growth Factor Beta 1/Mitogen-Activated Protein Kinase Signaling Pathway

    Journal: Chinese Medical Journal

    doi: 10.4103/0366-6999.238151

    FSTL1 modulates the activation of MAPK signaling in primary lung fibroblast. Western blotting analysis of total and phosphorylated ERK, p38, and JNK in lung tissues (a) and primary lung fibroblasts (b and c) from Fstl1 +/− and their WT littermates. Primary lung fibroblasts were treated with 5 ng/ml TGF-β1 and/or 100 ng/ml FSTL1 protein. β-Tubulin and GAPDH served as control loadings. n = 3; * P
    Figure Legend Snippet: FSTL1 modulates the activation of MAPK signaling in primary lung fibroblast. Western blotting analysis of total and phosphorylated ERK, p38, and JNK in lung tissues (a) and primary lung fibroblasts (b and c) from Fstl1 +/− and their WT littermates. Primary lung fibroblasts were treated with 5 ng/ml TGF-β1 and/or 100 ng/ml FSTL1 protein. β-Tubulin and GAPDH served as control loadings. n = 3; * P

    Techniques Used: Activation Assay, Western Blot

    FSTL1 modulates myofibroblast differentiation by facilitating p38/JNK signaling. (a and b) Primary lung fibroblasts from Fstl1+/− and their WT littermates were treated with 5 ng/ml TGF-β1. (a) Immunofluorescence staining of α-SMA in lung fibroblasts. (b) Protein expression levels of α-SMA and type I collagen. (c-e) MLgs were pretreated with U0126, SB202190, and SP600125. Protein expression levels of α-SMA and type I collagen were detected by Western blotting. n = 4; Bars = 100 μm. * P
    Figure Legend Snippet: FSTL1 modulates myofibroblast differentiation by facilitating p38/JNK signaling. (a and b) Primary lung fibroblasts from Fstl1+/− and their WT littermates were treated with 5 ng/ml TGF-β1. (a) Immunofluorescence staining of α-SMA in lung fibroblasts. (b) Protein expression levels of α-SMA and type I collagen. (c-e) MLgs were pretreated with U0126, SB202190, and SP600125. Protein expression levels of α-SMA and type I collagen were detected by Western blotting. n = 4; Bars = 100 μm. * P

    Techniques Used: Immunofluorescence, Staining, Expressing, Western Blot

    FSTL1 promotes fibroblasts proliferation, migration, and invasion through positively regulating p38/JNK/Smad2/3 signaling. (a) Cell proliferation was measured by MTT. MLgs migration (b) and invasion (c) were measured by transwell chambers. Representative histogram represents cells per field. n = 3, Bars = 100 μm. * P
    Figure Legend Snippet: FSTL1 promotes fibroblasts proliferation, migration, and invasion through positively regulating p38/JNK/Smad2/3 signaling. (a) Cell proliferation was measured by MTT. MLgs migration (b) and invasion (c) were measured by transwell chambers. Representative histogram represents cells per field. n = 3, Bars = 100 μm. * P

    Techniques Used: Migration, MTT Assay

    4) Product Images from "The ameliorative effect of hemp seed hexane extracts on the Propionibacterium acnes-induced inflammation and lipogenesis in sebocytes"

    Article Title: The ameliorative effect of hemp seed hexane extracts on the Propionibacterium acnes-induced inflammation and lipogenesis in sebocytes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0202933

    Hemp seed hexane extracts effectively inhibit the MAPK signaling pathway in P . acnes -treated HaCaT cells. (A) The expression of p-p38, p-ERK, and p-JNK were analyzed with ImageJ and normalized against β-actin. (B) Transcriptional activation of AP-1 was analyzed by luciferase reporter assay. AP-1 signaling was down-regulated by HSHE in HaCaT cells. Results are expressed as mean ± SD. *P
    Figure Legend Snippet: Hemp seed hexane extracts effectively inhibit the MAPK signaling pathway in P . acnes -treated HaCaT cells. (A) The expression of p-p38, p-ERK, and p-JNK were analyzed with ImageJ and normalized against β-actin. (B) Transcriptional activation of AP-1 was analyzed by luciferase reporter assay. AP-1 signaling was down-regulated by HSHE in HaCaT cells. Results are expressed as mean ± SD. *P

    Techniques Used: Expressing, Activation Assay, Luciferase, Reporter Assay

    5) Product Images from "Poligoni Multiflori Radix enhances osteoblast formation and reduces osteoclast differentiation"

    Article Title: Poligoni Multiflori Radix enhances osteoblast formation and reduces osteoclast differentiation

    Journal: International Journal of Molecular Medicine

    doi: 10.3892/ijmm.2018.3603

    Effect of PMR on osteoblast-specific markers and on the p38 and Smad pathways. (A) Primary mouse osteoblasts were seeded in 6-well plates at a density 2×10 5 cells/well and cultured in the absence or presence of β-GP (10 mM) and AA (50 µ g/ml) for the indicated days. Levels of mRNA expression of Runx2, ALP, osterix and osteocalcin were analyzed by reverse transcription-quantitative polymerase chain reaction. (B) Primary mouse osteoblasts were serum-starved for 2 h, and treated with PMR (50 µ g/ml) for the indicated time. The cell lysates were analyzed by western blotting with the indicated antibodies. β-actin was used as an internal control. * P
    Figure Legend Snippet: Effect of PMR on osteoblast-specific markers and on the p38 and Smad pathways. (A) Primary mouse osteoblasts were seeded in 6-well plates at a density 2×10 5 cells/well and cultured in the absence or presence of β-GP (10 mM) and AA (50 µ g/ml) for the indicated days. Levels of mRNA expression of Runx2, ALP, osterix and osteocalcin were analyzed by reverse transcription-quantitative polymerase chain reaction. (B) Primary mouse osteoblasts were serum-starved for 2 h, and treated with PMR (50 µ g/ml) for the indicated time. The cell lysates were analyzed by western blotting with the indicated antibodies. β-actin was used as an internal control. * P

    Techniques Used: Cell Culture, Expressing, ALP Assay, Real-time Polymerase Chain Reaction, Western Blot

    6) Product Images from "Chinese herb pair Paeoniae Radix Alba and Atractylodis Macrocephalae Rhizoma suppresses LPS-induced inflammatory response through inhibiting MAPK and NF-κB pathway"

    Article Title: Chinese herb pair Paeoniae Radix Alba and Atractylodis Macrocephalae Rhizoma suppresses LPS-induced inflammatory response through inhibiting MAPK and NF-κB pathway

    Journal: Chinese Medicine

    doi: 10.1186/s13020-019-0224-2

    OPAE decreased the phosphorylation of MAPKs in LPS-stimulated RAW 264.7 macrophages. Cells were pre-treated with OPAE at different concentrations (125, 250, and 500 mg mL −1 ) for 4 h and then stimulated with LPS (1 µg mL −1 ) for another 30 min. The protein levels of a ERK and p-ERK, b JNK and p-JNK, c p38 and p-p38 were determined by western blotting (n = 3), GAPDH was used as internal control. Data are expressed as mean ± S.D. ### p
    Figure Legend Snippet: OPAE decreased the phosphorylation of MAPKs in LPS-stimulated RAW 264.7 macrophages. Cells were pre-treated with OPAE at different concentrations (125, 250, and 500 mg mL −1 ) for 4 h and then stimulated with LPS (1 µg mL −1 ) for another 30 min. The protein levels of a ERK and p-ERK, b JNK and p-JNK, c p38 and p-p38 were determined by western blotting (n = 3), GAPDH was used as internal control. Data are expressed as mean ± S.D. ### p

    Techniques Used: Western Blot

    7) Product Images from "Kv1.3 modulates neuroinflammation and neurodegeneration in Parkinson’s disease"

    Article Title: Kv1.3 modulates neuroinflammation and neurodegeneration in Parkinson’s disease

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI136174

    Fyn modulates the transcriptional regulation of Kv1.3 in microglial cells through the Fyn/PKCδ kinase signaling cascade. ( A ) In silico analysis of the promoter sequence of Kv1.3 revealed probable Nf-κB– and SP1-binding sites. ( B ) qRT-PCR analysis of immortalized MMCs cotreated with αSyn Agg and either SN50 (100 μg/mL) or SB203580 (1 μM), showing that both compounds attenuated αSyn Agg -induced Kv1.3 expression. ( C ) Western blot of Fyn WT and KO PMCs treated with αSyn Agg , showing that Fyn KO reduced the induction of the p38 MAPK pathway. ( D ) qRT-PCR analysis revealed that Fyn KO reduced αSyn Agg -induced Kv1.3 mRNA levels. ( E ) Whole-cell patch-clamp recording showing that Fyn KO attenuated αSyn Agg - and LPS-induced Kv1.3 activity compared with Fyn WT PMCs (WT control n = 24, WT αSyn Agg n = 12, WT LPS n = 29, Fyn KO αSyn Agg n = 20, Fyn KO LPS n = 15). ( F ) ICC showing that Fyn KO reduced αSyn Agg -induced Kv1.3 protein levels in PMCs. Scale bar: 15 μm. ( G ) ICC of PMCs revealed that αSyn Agg -induced Kv1.3 protein expression was reduced by PKCδ KO. Scale bar: 15 μm. ( H ) qRT-PCR analysis of PMCs showing that PKC KO reduced the expression of αSyn Agg -induced Kv1.3 mRNA. ( I ) Whole-cell patch clam recording of PMCs showing that PKC KO attenuated αSyn Agg - and LPS-induced Kv1.3 activity compared with PKC WT PMCs (WT control n = 24, WT αSyn Agg n = 12, WT LPS n = 20, PKC-KO αSyn Agg n = 29, PKC-KO LPS n = 35). Data are presented as the mean ± SD. A 1-way ANOVA was used to compare multiple groups. In most cases, Tukey’s post hoc analysis was applied. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–4 biological replicates from 2–3 independent experiments unless otherwise indicated. * P ≤ 0.05, ** P
    Figure Legend Snippet: Fyn modulates the transcriptional regulation of Kv1.3 in microglial cells through the Fyn/PKCδ kinase signaling cascade. ( A ) In silico analysis of the promoter sequence of Kv1.3 revealed probable Nf-κB– and SP1-binding sites. ( B ) qRT-PCR analysis of immortalized MMCs cotreated with αSyn Agg and either SN50 (100 μg/mL) or SB203580 (1 μM), showing that both compounds attenuated αSyn Agg -induced Kv1.3 expression. ( C ) Western blot of Fyn WT and KO PMCs treated with αSyn Agg , showing that Fyn KO reduced the induction of the p38 MAPK pathway. ( D ) qRT-PCR analysis revealed that Fyn KO reduced αSyn Agg -induced Kv1.3 mRNA levels. ( E ) Whole-cell patch-clamp recording showing that Fyn KO attenuated αSyn Agg - and LPS-induced Kv1.3 activity compared with Fyn WT PMCs (WT control n = 24, WT αSyn Agg n = 12, WT LPS n = 29, Fyn KO αSyn Agg n = 20, Fyn KO LPS n = 15). ( F ) ICC showing that Fyn KO reduced αSyn Agg -induced Kv1.3 protein levels in PMCs. Scale bar: 15 μm. ( G ) ICC of PMCs revealed that αSyn Agg -induced Kv1.3 protein expression was reduced by PKCδ KO. Scale bar: 15 μm. ( H ) qRT-PCR analysis of PMCs showing that PKC KO reduced the expression of αSyn Agg -induced Kv1.3 mRNA. ( I ) Whole-cell patch clam recording of PMCs showing that PKC KO attenuated αSyn Agg - and LPS-induced Kv1.3 activity compared with PKC WT PMCs (WT control n = 24, WT αSyn Agg n = 12, WT LPS n = 20, PKC-KO αSyn Agg n = 29, PKC-KO LPS n = 35). Data are presented as the mean ± SD. A 1-way ANOVA was used to compare multiple groups. In most cases, Tukey’s post hoc analysis was applied. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–4 biological replicates from 2–3 independent experiments unless otherwise indicated. * P ≤ 0.05, ** P

    Techniques Used: In Silico, Sequencing, Binding Assay, Quantitative RT-PCR, Expressing, Western Blot, Patch Clamp, Activity Assay, Immunocytochemistry

    8) Product Images from "SOCS3 Suppression Promoted the Recruitment of CD11b+Gr-1−F4/80−MHCII− Early-Stage Myeloid-Derived Suppressor Cells and Accelerated Interleukin-6-Related Tumor Invasion via Affecting Myeloid Differentiation in Breast Cancer"

    Article Title: SOCS3 Suppression Promoted the Recruitment of CD11b+Gr-1−F4/80−MHCII− Early-Stage Myeloid-Derived Suppressor Cells and Accelerated Interleukin-6-Related Tumor Invasion via Affecting Myeloid Differentiation in Breast Cancer

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.01699

    Interleukin-6 (IL-6)-mediated suppressed expression of SOCS3 resulting in the hyperactivation of the JAK/STAT pathway in CD11b + Gr-1 − early-stage MDSCs (e-MDSCs). Western blotting was performed to detect the JAK/STAT (A) and MAPK (B) on the whole-cell extracts of primary CD11b + Gr-1 − e-MDSCs isolated from different IL-6-expressing tumors. (C) The protein expression of SOCS1–3 in primary CD11b + Gr-1 − e-MDSCs. (D) The mRNA of SOCS1–3 in primary CD11b + Gr-1 − e-MDSCs was examined by RT-PCR. (E) Bone marrow (BM) cells were treated with 4T1 NC and 4T1 IL-6low separately to induce iMDSCs NC and iMDSCs IL-6low . The activation status of the JAK/STAT pathway downstream of IL-6 signaling in induce MDSCs (iMDSCs) was detected at different time point. BM cells exposed to IL-6 (40 ng/mL) for 15 min were as control. The levels of phosphorylated JAK1 (p-JAK1), p-JAK2, p-TYK2, p-STAT1, p-STAT3, p-P38, p-ERK, and p-JNK were compared using the density ratio of phosphorylated protein to total protein. The levels of the suppressor of cytokine signaling (SOCS) protein were compared using the density ratio of the indicated protein to β-actin (* P
    Figure Legend Snippet: Interleukin-6 (IL-6)-mediated suppressed expression of SOCS3 resulting in the hyperactivation of the JAK/STAT pathway in CD11b + Gr-1 − early-stage MDSCs (e-MDSCs). Western blotting was performed to detect the JAK/STAT (A) and MAPK (B) on the whole-cell extracts of primary CD11b + Gr-1 − e-MDSCs isolated from different IL-6-expressing tumors. (C) The protein expression of SOCS1–3 in primary CD11b + Gr-1 − e-MDSCs. (D) The mRNA of SOCS1–3 in primary CD11b + Gr-1 − e-MDSCs was examined by RT-PCR. (E) Bone marrow (BM) cells were treated with 4T1 NC and 4T1 IL-6low separately to induce iMDSCs NC and iMDSCs IL-6low . The activation status of the JAK/STAT pathway downstream of IL-6 signaling in induce MDSCs (iMDSCs) was detected at different time point. BM cells exposed to IL-6 (40 ng/mL) for 15 min were as control. The levels of phosphorylated JAK1 (p-JAK1), p-JAK2, p-TYK2, p-STAT1, p-STAT3, p-P38, p-ERK, and p-JNK were compared using the density ratio of phosphorylated protein to total protein. The levels of the suppressor of cytokine signaling (SOCS) protein were compared using the density ratio of the indicated protein to β-actin (* P

    Techniques Used: Expressing, Western Blot, Isolation, Reverse Transcription Polymerase Chain Reaction, Activation Assay

    9) Product Images from "Myeloid Heme Oxygenase-1 Haploinsufficiency Reduces High Fat Diet-Induced Insulin Resistance by Affecting Adipose Macrophage Infiltration in Mice"

    Article Title: Myeloid Heme Oxygenase-1 Haploinsufficiency Reduces High Fat Diet-Induced Insulin Resistance by Affecting Adipose Macrophage Infiltration in Mice

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0038626

    CO promotes macrophage migration by modulating p38 signaling. A, Peritoneal macrophages from WT mice were tested in a transwell migration assay without or with MCP-1 (10 ng/ml). Macrophages in the upper chamber were concurrently treated without or with the indicated concentrations of CORM-2 or iCORM-2. The data are the mean ± SEM for three independent experiments. * P
    Figure Legend Snippet: CO promotes macrophage migration by modulating p38 signaling. A, Peritoneal macrophages from WT mice were tested in a transwell migration assay without or with MCP-1 (10 ng/ml). Macrophages in the upper chamber were concurrently treated without or with the indicated concentrations of CORM-2 or iCORM-2. The data are the mean ± SEM for three independent experiments. * P

    Techniques Used: Migration, Mouse Assay, Transwell Migration Assay

    10) Product Images from "Delphinidin induces apoptosis and inhibits epithelial‐to‐mesenchymal transition via the ERK/p38 MAPK‐signaling pathway in human osteosarcoma cell lines. Delphinidin induces apoptosis and inhibits epithelial‐to‐mesenchymal transition via the ERK/p38 MAPK‐signaling pathway in human osteosarcoma cell lines"

    Article Title: Delphinidin induces apoptosis and inhibits epithelial‐to‐mesenchymal transition via the ERK/p38 MAPK‐signaling pathway in human osteosarcoma cell lines. Delphinidin induces apoptosis and inhibits epithelial‐to‐mesenchymal transition via the ERK/p38 MAPK‐signaling pathway in human osteosarcoma cell lines

    Journal: Environmental Toxicology

    doi: 10.1002/tox.22548

    Inhibition of ERK and p38 suppressed the EMT effect of delphinidin in OS cells. (A,B) The OS cells were pretreated with an ERK1/2 and p38 inhibitor, and were detected using a western blot analysis. The levels of β‐actin were used as an internal standard for quantifying ERK, p38, and EMT factor expression
    Figure Legend Snippet: Inhibition of ERK and p38 suppressed the EMT effect of delphinidin in OS cells. (A,B) The OS cells were pretreated with an ERK1/2 and p38 inhibitor, and were detected using a western blot analysis. The levels of β‐actin were used as an internal standard for quantifying ERK, p38, and EMT factor expression

    Techniques Used: Inhibition, Western Blot, Expressing

    11) Product Images from "Reduced BMPR2 expression induces GM-CSF translation and macrophage recruitment in humans and mice to exacerbate pulmonary hypertension"

    Article Title: Reduced BMPR2 expression induces GM-CSF translation and macrophage recruitment in humans and mice to exacerbate pulmonary hypertension

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20111741

    Reduced BMPR2 induces sustained activation of p38 and MK-2. (A) Representative Western immunoblots for BMPR2, p-p38, total p38, p-MK2, total MK2, p–I-κB, and total I-κB in PAECs transfected with BMPR2 siRNA (“B”) or control siRNA (Con), 10, 30, and 60 min after stimulation with 10 ng/ml TNF. (B–D) Densitometric analysis of the immunoreactive bands for p38 (B), MK-2 (C), and I-κB (D). Bars are means ± SEM for n = 3 experiments. *, P
    Figure Legend Snippet: Reduced BMPR2 induces sustained activation of p38 and MK-2. (A) Representative Western immunoblots for BMPR2, p-p38, total p38, p-MK2, total MK2, p–I-κB, and total I-κB in PAECs transfected with BMPR2 siRNA (“B”) or control siRNA (Con), 10, 30, and 60 min after stimulation with 10 ng/ml TNF. (B–D) Densitometric analysis of the immunoreactive bands for p38 (B), MK-2 (C), and I-κB (D). Bars are means ± SEM for n = 3 experiments. *, P

    Techniques Used: Activation Assay, Western Blot, Transfection

    Reduced BMPR2 inhibits eIF2α phosphorylation in a p-p38– and PP1-dependent manner. (A) PAECs were transfected with BMPR2 (“B”) or control siRNA (Con) and harvested either before (0) or 30 and 60 min after TNF stimulation. Phosphorylation of the α subunit of eIF2α analyzed by Western immunoblot is shown above with densitometry below. Bars are mean ± SEM for n = 3 experiments. *, P
    Figure Legend Snippet: Reduced BMPR2 inhibits eIF2α phosphorylation in a p-p38– and PP1-dependent manner. (A) PAECs were transfected with BMPR2 (“B”) or control siRNA (Con) and harvested either before (0) or 30 and 60 min after TNF stimulation. Phosphorylation of the α subunit of eIF2α analyzed by Western immunoblot is shown above with densitometry below. Bars are mean ± SEM for n = 3 experiments. *, P

    Techniques Used: Transfection, Western Blot

    12) Product Images from "Infection with the dengue RNA virus activates TLR9 signaling in human dendritic cells"

    Article Title: Infection with the dengue RNA virus activates TLR9 signaling in human dendritic cells

    Journal: EMBO Reports

    doi: 10.15252/embr.201846182

    Introduction of mtDNA simulated DENV infection‐mediated effects in DCs A–E Both mtDNA and nDNA were prepared as described in the Materials and Methods. Either mtDNA or nDNA at 1 μg was delivered into DCs via electroporation. Null‐electroporated cells were used as controls. Several parameters, including IFN mRNA (A, n = 5 for IFN‐β1 and IFN‐λ1, n = 3 for IFN‐λ2 and n = 4 for IFN‐λ3), TLR9 mRNA (B, n = 4), and TLR9 protein expression (C, n = 9 for MFI measurement and n = 4 for percentages analysis), were measured. The levels of phosphorylated and unphosphorylated p65, p38, and IRF7 (D, n = 3) and expression of CCR7 mRNA (E, n = 5) were determined. DCs infected by mock or DENV (MOI = 5) for 24 h served for additional comparisons in (A). Values are means of individual measurements in each sample ± SEM. * P
    Figure Legend Snippet: Introduction of mtDNA simulated DENV infection‐mediated effects in DCs A–E Both mtDNA and nDNA were prepared as described in the Materials and Methods. Either mtDNA or nDNA at 1 μg was delivered into DCs via electroporation. Null‐electroporated cells were used as controls. Several parameters, including IFN mRNA (A, n = 5 for IFN‐β1 and IFN‐λ1, n = 3 for IFN‐λ2 and n = 4 for IFN‐λ3), TLR9 mRNA (B, n = 4), and TLR9 protein expression (C, n = 9 for MFI measurement and n = 4 for percentages analysis), were measured. The levels of phosphorylated and unphosphorylated p65, p38, and IRF7 (D, n = 3) and expression of CCR7 mRNA (E, n = 5) were determined. DCs infected by mock or DENV (MOI = 5) for 24 h served for additional comparisons in (A). Values are means of individual measurements in each sample ± SEM. * P

    Techniques Used: Infection, Electroporation, Expressing

    TLR9 activation contributed to DENV‐induced IFN production A–C Human DCs were transfected with control siRNA (Ctl) or three sets of TLR9 siRNA duplexes for 24 h and then infected with mock or DENV (MOI = 5) for additional 24 h. The TLR9 mRNA (A, n = 3) and protein expression (B, n = 4) were determined by qPCR and flow cytometry, respectively. The levels of phosphorylated and unphosphorylated p65 ( n = 5) and p38 ( n = 3) and TLR9 in total cell lysates were analyzed and the relative band intensity measured (C). Values are means of individual measurements in each sample ± SEM. * P
    Figure Legend Snippet: TLR9 activation contributed to DENV‐induced IFN production A–C Human DCs were transfected with control siRNA (Ctl) or three sets of TLR9 siRNA duplexes for 24 h and then infected with mock or DENV (MOI = 5) for additional 24 h. The TLR9 mRNA (A, n = 3) and protein expression (B, n = 4) were determined by qPCR and flow cytometry, respectively. The levels of phosphorylated and unphosphorylated p65 ( n = 5) and p38 ( n = 3) and TLR9 in total cell lysates were analyzed and the relative band intensity measured (C). Values are means of individual measurements in each sample ± SEM. * P

    Techniques Used: Activation Assay, Transfection, Infection, Expressing, Real-time Polymerase Chain Reaction, Flow Cytometry

    13) Product Images from "Heparin and LPS-induced COX-2 expression in airway cells: a link between its anti-inflammatory effects and GAG sulfation"

    Article Title: Heparin and LPS-induced COX-2 expression in airway cells: a link between its anti-inflammatory effects and GAG sulfation

    Journal: Experimental lung research

    doi: 10.3109/01902148.2015.1091053

    The effect of HMW heparin on phosphorylation of ERK1/2 (A), p38 (B), and NF-κB p65 (C) in LPS-treated H292 cells. Levels of phosphorylation were determined 30 mins after treatment. HMW heparin at 500 µg/mL significantly prevented LPS-induced
    Figure Legend Snippet: The effect of HMW heparin on phosphorylation of ERK1/2 (A), p38 (B), and NF-κB p65 (C) in LPS-treated H292 cells. Levels of phosphorylation were determined 30 mins after treatment. HMW heparin at 500 µg/mL significantly prevented LPS-induced

    Techniques Used:

    The effect of sulfation levels of heparin on phosphorylation of ERK1/2 (A), p38 (B), and NF-κB p65 (C). H292 cells were treated with either HMW heparin (500 µg/mL) or desulfated HMW heparin (500 µg/mL) in the presence of 10 µg/mL
    Figure Legend Snippet: The effect of sulfation levels of heparin on phosphorylation of ERK1/2 (A), p38 (B), and NF-κB p65 (C). H292 cells were treated with either HMW heparin (500 µg/mL) or desulfated HMW heparin (500 µg/mL) in the presence of 10 µg/mL

    Techniques Used:

    The effect of HMW heparin on phosphorylation of ERK1/2 (A), p38 (B), and NF-κB p65 (C) in LPS-treated HBE-1 normal human bronchial epithelial cells. Levels of phosphorylation were determined 30 mins after treatment. HMW heparin at 50 and 500 µg/mL
    Figure Legend Snippet: The effect of HMW heparin on phosphorylation of ERK1/2 (A), p38 (B), and NF-κB p65 (C) in LPS-treated HBE-1 normal human bronchial epithelial cells. Levels of phosphorylation were determined 30 mins after treatment. HMW heparin at 50 and 500 µg/mL

    Techniques Used:

    14) Product Images from "Spinacetin Suppresses the Mast Cell Activation and Passive Cutaneous Anaphylaxis in Mouse Model"

    Article Title: Spinacetin Suppresses the Mast Cell Activation and Passive Cutaneous Anaphylaxis in Mouse Model

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2018.00824

    Spinacetin suppresses LTC 4 generation, cPLA 2 translocation, and MAPKs phosphorylation. IgE-sensitized BMMCs were pre-incubated with indicated concentrations of spinacetin or Bay 61-3606 for 1 h, then stimulated with DNP-HSA for 15 min. (A) The culture medium was collected and measured for generation of LTC 4 . (B) Cytosolic and nuclear fractions were immunoblotted with antibody for phospho-cPLA 2 , and relative ratios of cytosolic p-cPLA 2 (C) , nuclear p-cPLA 2 (D) proteins were determined by analyzing immunoblot band intensities. (E) Cell lysates were immunoblotted for the phosphorylated and total forms of ERK1/2, JNK1/2, and p38. Relative ratios of cytosolic p-ERK1/2/ERK1/2 (F) , p-JNK1/2/JNK1/2 (G) , and p-p38/p38 (H) proteins were determined by analyzing immunoblot band intensities. The results show the mean ± SEM of three independent experiments. ∗ P
    Figure Legend Snippet: Spinacetin suppresses LTC 4 generation, cPLA 2 translocation, and MAPKs phosphorylation. IgE-sensitized BMMCs were pre-incubated with indicated concentrations of spinacetin or Bay 61-3606 for 1 h, then stimulated with DNP-HSA for 15 min. (A) The culture medium was collected and measured for generation of LTC 4 . (B) Cytosolic and nuclear fractions were immunoblotted with antibody for phospho-cPLA 2 , and relative ratios of cytosolic p-cPLA 2 (C) , nuclear p-cPLA 2 (D) proteins were determined by analyzing immunoblot band intensities. (E) Cell lysates were immunoblotted for the phosphorylated and total forms of ERK1/2, JNK1/2, and p38. Relative ratios of cytosolic p-ERK1/2/ERK1/2 (F) , p-JNK1/2/JNK1/2 (G) , and p-p38/p38 (H) proteins were determined by analyzing immunoblot band intensities. The results show the mean ± SEM of three independent experiments. ∗ P

    Techniques Used: Translocation Assay, Incubation

    Spinacetin attenuates activation of RBL-2H3 without effect on HMC-1 cells. IgE-sensitized RBL-2H3 cells were pre-incubated with spinacetin for 1 h and then stimulated with DNP-HSA for 15 min. Cell lysates were immunoblotted for the phosphorylated and total forms of ERK1/2, JNK1/2, p38, and cytosolic and nuclear p-cPLA 2 (A,D) , p-Akt, p-IκBα, IκBα, cytosolic and nuclear NF-κB p65 (B,E) , and p-PLCγ (C,F) . Relative intensities of the respective signal proteins were determined by using ImageJ software. The results show the mean ± SEM of three independent experiments. “–”: RBL-2H3 cells sensitized with IgE; “+”: RBL-2H3 cells sensitized with IgE/DNP-HSA. Bay: Bay 61-3606. (G) HMC-1 cells were pre-treated with spinacetin for 1 h, then stimulated with PMA plus A23187 for 15 min. HMC-1 cells lysates were prepared for Western blot of p-ERK1/2, p-JNK1/2, p-p38, p-cPLA 2 , p-Akt, and IKKα/β proteins. (H) Relative intensities of the respective signal proteins in (G) were determined by using ImageJ software. The results show the mean ± SEM of three independent experiments. “–”: non-treated HMC-1 cells; “+”: HMC-1 cells sensitized with PMA/A23187. ∗ P
    Figure Legend Snippet: Spinacetin attenuates activation of RBL-2H3 without effect on HMC-1 cells. IgE-sensitized RBL-2H3 cells were pre-incubated with spinacetin for 1 h and then stimulated with DNP-HSA for 15 min. Cell lysates were immunoblotted for the phosphorylated and total forms of ERK1/2, JNK1/2, p38, and cytosolic and nuclear p-cPLA 2 (A,D) , p-Akt, p-IκBα, IκBα, cytosolic and nuclear NF-κB p65 (B,E) , and p-PLCγ (C,F) . Relative intensities of the respective signal proteins were determined by using ImageJ software. The results show the mean ± SEM of three independent experiments. “–”: RBL-2H3 cells sensitized with IgE; “+”: RBL-2H3 cells sensitized with IgE/DNP-HSA. Bay: Bay 61-3606. (G) HMC-1 cells were pre-treated with spinacetin for 1 h, then stimulated with PMA plus A23187 for 15 min. HMC-1 cells lysates were prepared for Western blot of p-ERK1/2, p-JNK1/2, p-p38, p-cPLA 2 , p-Akt, and IKKα/β proteins. (H) Relative intensities of the respective signal proteins in (G) were determined by using ImageJ software. The results show the mean ± SEM of three independent experiments. “–”: non-treated HMC-1 cells; “+”: HMC-1 cells sensitized with PMA/A23187. ∗ P

    Techniques Used: Activation Assay, Incubation, Software, Western Blot

    15) Product Images from "Induction of Proinflammatory Responses in Macrophages by the Glycosylphosphatidylinositols (GPIs) of Plasmodium falciparum: The requirement of ERK, p38, JNK and NF-κB pathways for the expression of proinflammatory cytokines and nitric oxide"

    Article Title: Induction of Proinflammatory Responses in Macrophages by the Glycosylphosphatidylinositols (GPIs) of Plasmodium falciparum: The requirement of ERK, p38, JNK and NF-κB pathways for the expression of proinflammatory cytokines and nitric oxide

    Journal: The Journal of biological chemistry

    doi: 10.1074/jbc.M413539200

    The effect of p38 inhibitor on the production of inflammatory cytokines and nitric oxide in macrophages stimulated with P. falciparum GPIs Panel A : Bone marrow-derived macrophages from C57BL6/J mice were plated in 96-well microtiter plates (2.5 × 10 4 cells/well). After culturing overnight, the cells were treated with the indicated amounts of SB203580 for 1 h. The cells were primed with IFNγ and then stimulated with 40 nM GPIs in the continued presence of the inhibitor. Cells primed and stimulated with GPIs but not pretreated with the inhibitors were used as controls. After 48 h stimulation, the culture supernatants were assayed for TNF-α, IL-6, IL-12, and nitric oxide. Panel B : Bone marrow-derived macrophages in 24-well microtiter plates (5 × 10 5 cells/well) were treated with SB203580 and then stimulated with 200 nM GPIs for 25 min. The cell lysates analyzed by Western blotting using anti-p38 peptide and phospho-specific antibodies.
    Figure Legend Snippet: The effect of p38 inhibitor on the production of inflammatory cytokines and nitric oxide in macrophages stimulated with P. falciparum GPIs Panel A : Bone marrow-derived macrophages from C57BL6/J mice were plated in 96-well microtiter plates (2.5 × 10 4 cells/well). After culturing overnight, the cells were treated with the indicated amounts of SB203580 for 1 h. The cells were primed with IFNγ and then stimulated with 40 nM GPIs in the continued presence of the inhibitor. Cells primed and stimulated with GPIs but not pretreated with the inhibitors were used as controls. After 48 h stimulation, the culture supernatants were assayed for TNF-α, IL-6, IL-12, and nitric oxide. Panel B : Bone marrow-derived macrophages in 24-well microtiter plates (5 × 10 5 cells/well) were treated with SB203580 and then stimulated with 200 nM GPIs for 25 min. The cell lysates analyzed by Western blotting using anti-p38 peptide and phospho-specific antibodies.

    Techniques Used: Derivative Assay, Mouse Assay, Western Blot

    16) Product Images from "Tuberatolide B Suppresses Cancer Progression by Promoting ROS-Mediated Inhibition of STAT3 Signaling"

    Article Title: Tuberatolide B Suppresses Cancer Progression by Promoting ROS-Mediated Inhibition of STAT3 Signaling

    Journal: Marine Drugs

    doi: 10.3390/md15030055

    TTB selectively suppresses STAT3 signaling pathway: ( A ) MDA-MB-231, A549 and HCT116 cells were treated with TTB (100 μM) for 15 min and whole lysates were analyzed by western blot with anti-p-EGFR (Y992), -p-EGFR (Y1068), -p-EGFR (Y1173), -EGFR, -p-AKT, -AKT, -p-ERK, -ERK, -p-JNK, -JNK, -p-p38, -p38, -p-STAT3 and -STAT3. Actin was used for internal control. Quantitative analyses of p-STAT3 expression was performed using the Image J software; ( B ) MDA-MB-231 cells were transfected with the STAT3-dependent luciferase reporter and then treated with TTB (100 μM) for 6 h. Luciferase assay were done by using dual-luciferase reporter assays. All transfections included the RLTK-Luc for transfection efficiency. * p
    Figure Legend Snippet: TTB selectively suppresses STAT3 signaling pathway: ( A ) MDA-MB-231, A549 and HCT116 cells were treated with TTB (100 μM) for 15 min and whole lysates were analyzed by western blot with anti-p-EGFR (Y992), -p-EGFR (Y1068), -p-EGFR (Y1173), -EGFR, -p-AKT, -AKT, -p-ERK, -ERK, -p-JNK, -JNK, -p-p38, -p38, -p-STAT3 and -STAT3. Actin was used for internal control. Quantitative analyses of p-STAT3 expression was performed using the Image J software; ( B ) MDA-MB-231 cells were transfected with the STAT3-dependent luciferase reporter and then treated with TTB (100 μM) for 6 h. Luciferase assay were done by using dual-luciferase reporter assays. All transfections included the RLTK-Luc for transfection efficiency. * p

    Techniques Used: Multiple Displacement Amplification, Western Blot, Expressing, Software, Transfection, Luciferase

    17) Product Images from "Anti-Inflammatory Effects of Fermented Lotus Root and Linoleic Acid in Lipopolysaccharide-Induced RAW 264.7 Cells"

    Article Title: Anti-Inflammatory Effects of Fermented Lotus Root and Linoleic Acid in Lipopolysaccharide-Induced RAW 264.7 Cells

    Journal: Life

    doi: 10.3390/life10110293

    Effects of LA on phosphorylation of extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK). RAW 264.7 cells were co-treated with LPS (1 μg/mL) and 1 to 100 μM LA for 1 h. Western blot analysis of phosphorylated and unphosphorylated ERK, p38, and JNK proteins to evaluate MAPK activation The images above are representatives of triplicate experiments. The data represent mean ± SEM, n = 3. * p
    Figure Legend Snippet: Effects of LA on phosphorylation of extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK). RAW 264.7 cells were co-treated with LPS (1 μg/mL) and 1 to 100 μM LA for 1 h. Western blot analysis of phosphorylated and unphosphorylated ERK, p38, and JNK proteins to evaluate MAPK activation The images above are representatives of triplicate experiments. The data represent mean ± SEM, n = 3. * p

    Techniques Used: Western Blot, Activation Assay

    18) Product Images from "HMGB1 promotes differentiation syndrome by inducing hyperinflammation via MEK/ERK signaling in acute promyelocytic leukemia cells"

    Article Title: HMGB1 promotes differentiation syndrome by inducing hyperinflammation via MEK/ERK signaling in acute promyelocytic leukemia cells

    Journal: Oncotarget

    doi: 10.18632/oncotarget.15432

    Role of MEK/ERK pathway in HMGB1-mediated cytokine secretion and ICAM-1 elevation A . The MAPK signaling pathway was analyzed in NB4 cells that were treated with ATRA (1 μM) for 48 h or/and HMGB1 (10 μg/ml) for 8 h by western blot analysis of phosphorylated and non-phosphorylated p38, Jnk and Erk kinases. Also, NF-kB pathway was analyzed by detecting the levels of p65 and IkB-α proteins for the same treatments described above along with PCNA. Actin was used as control. B . ICAM-1 levels were determined by FACS analysis of NB4 cells that were treated with ATRA (1 μM) for 48 h followed by HMGB1 (10 μg/ml) for 8 h with or without pre-treatment of the p38, Jnk, NF-kB or MEK inhibitors for 30 min (n=3, * P
    Figure Legend Snippet: Role of MEK/ERK pathway in HMGB1-mediated cytokine secretion and ICAM-1 elevation A . The MAPK signaling pathway was analyzed in NB4 cells that were treated with ATRA (1 μM) for 48 h or/and HMGB1 (10 μg/ml) for 8 h by western blot analysis of phosphorylated and non-phosphorylated p38, Jnk and Erk kinases. Also, NF-kB pathway was analyzed by detecting the levels of p65 and IkB-α proteins for the same treatments described above along with PCNA. Actin was used as control. B . ICAM-1 levels were determined by FACS analysis of NB4 cells that were treated with ATRA (1 μM) for 48 h followed by HMGB1 (10 μg/ml) for 8 h with or without pre-treatment of the p38, Jnk, NF-kB or MEK inhibitors for 30 min (n=3, * P

    Techniques Used: Western Blot, FACS

    19) Product Images from "Enhanced transforming growth factor-? signaling and fibrogenesis in ovine fetal skeletal muscle of obese dams at late gestation"

    Article Title: Enhanced transforming growth factor-? signaling and fibrogenesis in ovine fetal skeletal muscle of obese dams at late gestation

    Journal: American Journal of Physiology - Endocrinology and Metabolism

    doi: 10.1152/ajpendo.00015.2010

    TGFβ/p38 signaling in fetal St muscle of Con (open bars) and OB (filled bars) sheep. An increase in p38 phosphorylation was observed in OB compared with Con fetal muscle. * P
    Figure Legend Snippet: TGFβ/p38 signaling in fetal St muscle of Con (open bars) and OB (filled bars) sheep. An increase in p38 phosphorylation was observed in OB compared with Con fetal muscle. * P

    Techniques Used:

    20) Product Images from "Therapeutic Effect and Mechanism of Bushen-Jianpi-Jiedu Decoction Combined with Chemotherapeutic Drugs on Postoperative Colorectal Cancer"

    Article Title: Therapeutic Effect and Mechanism of Bushen-Jianpi-Jiedu Decoction Combined with Chemotherapeutic Drugs on Postoperative Colorectal Cancer

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2021.524663

    Expressions of MAPK pathway-related proteins treated by BSJPJDD and oxapilation in SW620 cells. (A) Fragments of Western Blot; (B–G) Expression of proteins (p-ERK,ERK,p38,p-p38,JNK,P-JNK) was quantified by ImageJ and normalized by GAPDH expression. Values represent the mean ± SD from three independent experiments * p
    Figure Legend Snippet: Expressions of MAPK pathway-related proteins treated by BSJPJDD and oxapilation in SW620 cells. (A) Fragments of Western Blot; (B–G) Expression of proteins (p-ERK,ERK,p38,p-p38,JNK,P-JNK) was quantified by ImageJ and normalized by GAPDH expression. Values represent the mean ± SD from three independent experiments * p

    Techniques Used: Western Blot, Expressing

    21) Product Images from "Inhibitors of Oxidative Phosphorylation Modulate Astrocyte Inflammatory Responses through AMPK-Dependent Ptgs2 mRNA Stabilization"

    Article Title: Inhibitors of Oxidative Phosphorylation Modulate Astrocyte Inflammatory Responses through AMPK-Dependent Ptgs2 mRNA Stabilization

    Journal: Cells

    doi: 10.3390/cells8101185

    Inhibitors of oxidative phosphorylation increase Ptgs2 mRNA stability. ( A ) Western blot analysis of c-Jun N-terminal kinase (JNK), IκB kinase (IKK), and p38 mitogen-activated protein kinase (MAPK) phosphorylation in astrocytes treated for 0.5 hours with IL-1α in the presence or absence of rotenone, oligomycin, and antimycin, as indicated. ( B ) The mRNA expression of Ptgs2 in astrocytes treated with IL-1α, antimycin, and oligomycin for 1 hour followed by actinomycin D for indicated times. Note: *, p
    Figure Legend Snippet: Inhibitors of oxidative phosphorylation increase Ptgs2 mRNA stability. ( A ) Western blot analysis of c-Jun N-terminal kinase (JNK), IκB kinase (IKK), and p38 mitogen-activated protein kinase (MAPK) phosphorylation in astrocytes treated for 0.5 hours with IL-1α in the presence or absence of rotenone, oligomycin, and antimycin, as indicated. ( B ) The mRNA expression of Ptgs2 in astrocytes treated with IL-1α, antimycin, and oligomycin for 1 hour followed by actinomycin D for indicated times. Note: *, p

    Techniques Used: Western Blot, Expressing

    22) Product Images from "A Physical Interaction Between the Adaptor Proteins DOK3 and DAP12 Is Required to Inhibit Lipopolysaccharide Signaling in Macrophages"

    Article Title: A Physical Interaction Between the Adaptor Proteins DOK3 and DAP12 Is Required to Inhibit Lipopolysaccharide Signaling in Macrophages

    Journal: Science signaling

    doi: 10.1126/scisignal.2003801

    DOK3 KO macrophages exhibit enhanced ERK phosphorylation in response to low-dose LPS ( A and B ) WT and DOK3 KO BMDMs were stimulated with LPS (1 ng/ml) for the indicated times. (A) Left: Cell lysates were analyzed by Western blotting for total and phosphorylated forms of ERK, JNK, and p38. β-Actin was used as a loading control. Right: Bar graph shows the densitometric analysis of Western blots from three independent experiments showing the mean fold change ± SD in the abundance of pERK relative to that of total ERK normalized to time zero. Statistical analysis was by two-way analysis of variance (ANOVA). (B) Western blotting analysis of IκBα abundance in lysates of WT and DOK3 KO BMDMs after stimulation with LPS (1 ng/ml) for the indicated times. β-Actin served as a loading control. Data are representative of three independent experiments.
    Figure Legend Snippet: DOK3 KO macrophages exhibit enhanced ERK phosphorylation in response to low-dose LPS ( A and B ) WT and DOK3 KO BMDMs were stimulated with LPS (1 ng/ml) for the indicated times. (A) Left: Cell lysates were analyzed by Western blotting for total and phosphorylated forms of ERK, JNK, and p38. β-Actin was used as a loading control. Right: Bar graph shows the densitometric analysis of Western blots from three independent experiments showing the mean fold change ± SD in the abundance of pERK relative to that of total ERK normalized to time zero. Statistical analysis was by two-way analysis of variance (ANOVA). (B) Western blotting analysis of IκBα abundance in lysates of WT and DOK3 KO BMDMs after stimulation with LPS (1 ng/ml) for the indicated times. β-Actin served as a loading control. Data are representative of three independent experiments.

    Techniques Used: Western Blot

    23) Product Images from "Immunotoxicological Effects of Aripiprazole: In vivo and In vitro Studies"

    Article Title: Immunotoxicological Effects of Aripiprazole: In vivo and In vitro Studies

    Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

    doi: 10.4196/kjpp.2015.19.4.365

    Effect of ARPGCB or GCB3004 on the level of cell death-related molecules in organs. (A, B, and C) The levels of cell death-related proteins such as (p65/NF-κB, c-Jun/AP-1, ERK/MAPK, JNK/MAPK, p38/MAPK, caspase 3, bcl-2, and β-actin) were identified by immunoblotting from tissue lysates prepared from mice orally administered with ARPGCB or GCB3004 (each of 500 mg/kg). The results shown are representative of three independent experiments.
    Figure Legend Snippet: Effect of ARPGCB or GCB3004 on the level of cell death-related molecules in organs. (A, B, and C) The levels of cell death-related proteins such as (p65/NF-κB, c-Jun/AP-1, ERK/MAPK, JNK/MAPK, p38/MAPK, caspase 3, bcl-2, and β-actin) were identified by immunoblotting from tissue lysates prepared from mice orally administered with ARPGCB or GCB3004 (each of 500 mg/kg). The results shown are representative of three independent experiments.

    Techniques Used: Mouse Assay

    24) Product Images from "Cordycepin inhibits LPS-induced inflammatory and matrix degradation in the intervertebral disc"

    Article Title: Cordycepin inhibits LPS-induced inflammatory and matrix degradation in the intervertebral disc

    Journal: PeerJ

    doi: 10.7717/peerj.1992

    Effect of cordycepin on the LPS-induced activation of the NF- κ B and MAPK pathways. NP cells were pretreated with various concentrations of cordycepin for 2 h and then stimulated with 10 µg/ml LPS for 24 h. Then, western blotting was performed to evaluate the mechanism of cordycepin on LPS-treated NP cells. (A) Cordycepin significantly inhibied the phosphorylation of I κ B α and p65 induced by LPS. (B) Cordycepin did not influence the phosphorylation of ERK, p38 or JNK enhanced by LPS. (C) NP cells were transfected with a NF- κ B luciferase reporter, and the NF- κ B pathway activity was determined by luciferase assay using a commercially available kit. (D–H) Quantitative analysis of western blotting data show cordycepin significantly inhibited the activation of the NF- κ B pathway induced by LPS at concentrations of 50 and 100 µM. The values are presented as the mean ± standard deviation. * P
    Figure Legend Snippet: Effect of cordycepin on the LPS-induced activation of the NF- κ B and MAPK pathways. NP cells were pretreated with various concentrations of cordycepin for 2 h and then stimulated with 10 µg/ml LPS for 24 h. Then, western blotting was performed to evaluate the mechanism of cordycepin on LPS-treated NP cells. (A) Cordycepin significantly inhibied the phosphorylation of I κ B α and p65 induced by LPS. (B) Cordycepin did not influence the phosphorylation of ERK, p38 or JNK enhanced by LPS. (C) NP cells were transfected with a NF- κ B luciferase reporter, and the NF- κ B pathway activity was determined by luciferase assay using a commercially available kit. (D–H) Quantitative analysis of western blotting data show cordycepin significantly inhibited the activation of the NF- κ B pathway induced by LPS at concentrations of 50 and 100 µM. The values are presented as the mean ± standard deviation. * P

    Techniques Used: Activation Assay, Western Blot, Transfection, Luciferase, Activity Assay, Standard Deviation

    25) Product Images from "JNK and p38 mitogen-activated protein kinase pathways contribute to porcine epidemic diarrhea virus infection"

    Article Title: JNK and p38 mitogen-activated protein kinase pathways contribute to porcine epidemic diarrhea virus infection

    Journal: Virus Research

    doi: 10.1016/j.virusres.2016.05.018

    Activation of p38 MAPK and JNK is required at early stages of PEDV infection but does not affect virus internalization. (A) Vero cells were pretreated with DMSO, SB202190 (5 and 10 μM), or SP600125 (1 and 5 μM) and were mock or PEDV (MOI of 1) infected. At the indicated time points post-infection, each inhibitor was added to attain the intended final concentration. At 48 hpi, the virus-infected cells were fixed, and virus infectivity was determined by measuring the percentage of cells expressing N proteins through FACS. (B) Virus internalization assay. Vero cells were infected at an MOI of 1 at 4 °C for 1 h. After washing with cold PBS, the infected cells were incubated with or without each inhibitor either at 4 °C or 37 °C for an additional hour. Bound but non-internalized viral particles were removed by treatment with proteinase K. The infected cells were then serially diluted and plated onto Vero cells in 96-well tissue culture plates. At 2 days post-incubation, internalized viruses were titrated by a plaque assay. The results are expressed as the mean values from triplicate wells, and the error bars represent standard deviations. *, P
    Figure Legend Snippet: Activation of p38 MAPK and JNK is required at early stages of PEDV infection but does not affect virus internalization. (A) Vero cells were pretreated with DMSO, SB202190 (5 and 10 μM), or SP600125 (1 and 5 μM) and were mock or PEDV (MOI of 1) infected. At the indicated time points post-infection, each inhibitor was added to attain the intended final concentration. At 48 hpi, the virus-infected cells were fixed, and virus infectivity was determined by measuring the percentage of cells expressing N proteins through FACS. (B) Virus internalization assay. Vero cells were infected at an MOI of 1 at 4 °C for 1 h. After washing with cold PBS, the infected cells were incubated with or without each inhibitor either at 4 °C or 37 °C for an additional hour. Bound but non-internalized viral particles were removed by treatment with proteinase K. The infected cells were then serially diluted and plated onto Vero cells in 96-well tissue culture plates. At 2 days post-incubation, internalized viruses were titrated by a plaque assay. The results are expressed as the mean values from triplicate wells, and the error bars represent standard deviations. *, P

    Techniques Used: Activation Assay, Infection, Concentration Assay, Expressing, FACS, Incubation, Plaque Assay

    PEDV propagation is suppressed by inhibition of p38 MAPK and JNK1/2 activation. (A) Vero cells were preincubated with DMSO, SB202190 (5 and 10 μM), or SP600125 (1 and 5 μM) for 1 h prior to infection and were mock-infected or infected with PEDV at an MOI of 1. The virus-infected cells were further maintained for 48 h in the presence of DMSO or inhibitors. PEDV-specific CPEs were monitored daily and were photographed at 48 hpi under an inverted microscope at the magnification of 100× (first panels). For immunostaining, the infected cells were fixed at 48 hpi and incubated with a MAb against the N protein followed by Alexa green-conjugated goat anti-mouse secondary antibody (second panels). The cells were then counterstained with DAPI (third panels) and examined using a fluorescent microscope at 200× magnification. (B) Viral production in the presence of each inhibitor was calculated by measuring the percentage of cells expressing N proteins through flow cytometry. The values shown are representative of three independent experiments, and the error bars represent standard deviations. **, P
    Figure Legend Snippet: PEDV propagation is suppressed by inhibition of p38 MAPK and JNK1/2 activation. (A) Vero cells were preincubated with DMSO, SB202190 (5 and 10 μM), or SP600125 (1 and 5 μM) for 1 h prior to infection and were mock-infected or infected with PEDV at an MOI of 1. The virus-infected cells were further maintained for 48 h in the presence of DMSO or inhibitors. PEDV-specific CPEs were monitored daily and were photographed at 48 hpi under an inverted microscope at the magnification of 100× (first panels). For immunostaining, the infected cells were fixed at 48 hpi and incubated with a MAb against the N protein followed by Alexa green-conjugated goat anti-mouse secondary antibody (second panels). The cells were then counterstained with DAPI (third panels) and examined using a fluorescent microscope at 200× magnification. (B) Viral production in the presence of each inhibitor was calculated by measuring the percentage of cells expressing N proteins through flow cytometry. The values shown are representative of three independent experiments, and the error bars represent standard deviations. **, P

    Techniques Used: Inhibition, Activation Assay, Infection, Inverted Microscopy, Immunostaining, Incubation, Microscopy, Expressing, Flow Cytometry

    PEDV activates the p38 MAPK and JNK1/2 signaling pathways in cultured cells. Vero cells were mock infected or infected with PEDV at an MOI of 1 (A and C) or an equal amount of UV-inactivated PEDV (B and D). Whole cell lysates were prepared for the indicated time points following PEDV infection and subjected to western blot analysis with the antibody specific for phosphorylated p38 (p-p38), p38, p-JNK1/2, JNK1/2, or the PEDV N protein. The blot was also reacted with a mouse MAb against b-actin to verify equal protein loading. Fold changes in ratios of p-p38:total p38 and p-JNK1/2:total JNK1/2 compared by densitometry of the corresponding bands using a computer densitometer are plotted. These data are representative of the results of three independent experiments and error bars represent standard deviations. *, P
    Figure Legend Snippet: PEDV activates the p38 MAPK and JNK1/2 signaling pathways in cultured cells. Vero cells were mock infected or infected with PEDV at an MOI of 1 (A and C) or an equal amount of UV-inactivated PEDV (B and D). Whole cell lysates were prepared for the indicated time points following PEDV infection and subjected to western blot analysis with the antibody specific for phosphorylated p38 (p-p38), p38, p-JNK1/2, JNK1/2, or the PEDV N protein. The blot was also reacted with a mouse MAb against b-actin to verify equal protein loading. Fold changes in ratios of p-p38:total p38 and p-JNK1/2:total JNK1/2 compared by densitometry of the corresponding bands using a computer densitometer are plotted. These data are representative of the results of three independent experiments and error bars represent standard deviations. *, P

    Techniques Used: Cell Culture, Infection, Western Blot

    Pharmacological inhibition of p38 MAPK and JNK activation interferes with viral RNA synthesis. Vero cells pretreated with DMSO, SB202190, or SP600125 were mock-infected or infected with PEDV (MOI of 1) for 1 h and were incubated with either DMSO or each inhibitor. (A) Total cellular RNA was extracted at 48 hpi, and strand-specific viral genomic RNA (black bars) and sg mRNA (white bars) were amplified by quantitative real-time RT-PCR. Viral positive-sense genomic RNA and sg mRNA were normalized to mRNA of monkey GAPDH, and relative quantities (RQ) of mRNA accumulation were evaluated. The results on inhibitor-treated sample were compared with DMSO-treated results. The values shown are representative of the mean from three independent experiments, and the error bars denote standard deviations. *, P
    Figure Legend Snippet: Pharmacological inhibition of p38 MAPK and JNK activation interferes with viral RNA synthesis. Vero cells pretreated with DMSO, SB202190, or SP600125 were mock-infected or infected with PEDV (MOI of 1) for 1 h and were incubated with either DMSO or each inhibitor. (A) Total cellular RNA was extracted at 48 hpi, and strand-specific viral genomic RNA (black bars) and sg mRNA (white bars) were amplified by quantitative real-time RT-PCR. Viral positive-sense genomic RNA and sg mRNA were normalized to mRNA of monkey GAPDH, and relative quantities (RQ) of mRNA accumulation were evaluated. The results on inhibitor-treated sample were compared with DMSO-treated results. The values shown are representative of the mean from three independent experiments, and the error bars denote standard deviations. *, P

    Techniques Used: Inhibition, Activation Assay, Infection, Incubation, Amplification, Quantitative RT-PCR

    Pharmacological inhibition of p38 MAPK and JNK activation impairs viral protein translation. Inhibitor-treated Vero cells were mock-infected or infected with PEDV (MOI of 1) for 1 h and were then cultivated in the presence or absence of each inhibitor. (A) At 48 hpi, cellular lysates were prepared, resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and immunoblotted with the antibody against the PEDV N protein. The blot was also reacted with a mouse MAb against β-actin to verify equal protein loading. Each viral protein expression was quantitatively analyzed by densitometry in terms of the density value relative to the β-actin gene, and inhibitor-treated sample results were compared to DMSO-control results. (B) The cells were also subjected to FACS analysis using the anti-PEDV N antibody to determine the mean fluorescence intensity for viral protein expression. The values shown are representative of the mean from three independent experiments, and the error bars denote standard deviations. *, P
    Figure Legend Snippet: Pharmacological inhibition of p38 MAPK and JNK activation impairs viral protein translation. Inhibitor-treated Vero cells were mock-infected or infected with PEDV (MOI of 1) for 1 h and were then cultivated in the presence or absence of each inhibitor. (A) At 48 hpi, cellular lysates were prepared, resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and immunoblotted with the antibody against the PEDV N protein. The blot was also reacted with a mouse MAb against β-actin to verify equal protein loading. Each viral protein expression was quantitatively analyzed by densitometry in terms of the density value relative to the β-actin gene, and inhibitor-treated sample results were compared to DMSO-control results. (B) The cells were also subjected to FACS analysis using the anti-PEDV N antibody to determine the mean fluorescence intensity for viral protein expression. The values shown are representative of the mean from three independent experiments, and the error bars denote standard deviations. *, P

    Techniques Used: Inhibition, Activation Assay, Infection, SDS Page, Expressing, FACS, Fluorescence

    26) Product Images from "CCL20 induced by visfatin in macrophages via the NF-κB and MKK3/6-p38 signaling pathways contributes to hepatic stellate cell activation"

    Article Title: CCL20 induced by visfatin in macrophages via the NF-κB and MKK3/6-p38 signaling pathways contributes to hepatic stellate cell activation

    Journal: Molecular Biology Reports

    doi: 10.1007/s11033-020-05510-7

    Visfatin induced activation of the NF-κB and MKK3/6-p38 MAPK signaling pathways in THP-1 cells. THP-1 cells were incubated with 200 ng/mL visfatin for the indicated times . a, b IKK/NF-κB signaling was analyzed using anti-phospho-IKKα/β and -phospho-NF-κB antibodies. JAK/STAT3 signaling was analyzed using anti-phospho-JAK2, -phospho-STAT3, and β-actin antibodies. c, d MAP kinase signaling was analyzed using anti-phospho-p38, -phospho-JNK, -phospho-ERK, and β-actin antibodies. e, f The MAPK signaling pathway consisting of MKK3/6 was analyzed using anti-phospho-MKK3/6 and β-actin antibodies. *p
    Figure Legend Snippet: Visfatin induced activation of the NF-κB and MKK3/6-p38 MAPK signaling pathways in THP-1 cells. THP-1 cells were incubated with 200 ng/mL visfatin for the indicated times . a, b IKK/NF-κB signaling was analyzed using anti-phospho-IKKα/β and -phospho-NF-κB antibodies. JAK/STAT3 signaling was analyzed using anti-phospho-JAK2, -phospho-STAT3, and β-actin antibodies. c, d MAP kinase signaling was analyzed using anti-phospho-p38, -phospho-JNK, -phospho-ERK, and β-actin antibodies. e, f The MAPK signaling pathway consisting of MKK3/6 was analyzed using anti-phospho-MKK3/6 and β-actin antibodies. *p

    Techniques Used: Activation Assay, Incubation

    Effects of signaling inhibitors on visfatin-induced CCL20 expression and release. THP-1 cells were pre-incubated with 5 µM BAY11-7082 (NF-κB inhibitor), 1 µM URMC-099 (MLK3 inhibitor), 10 µM SB 203,580 (p38 MAPK inhibitor), and 10 µM SP 600,125 (JNK inhibitor) for 4 h, and stimulated with visfatin (vis) for 20 h. a Expression of CCL20 was analyzed by real-time PCR. b CCL20 level in culture supernatants were measured by ELISA. Data are means ± standard errors of three independent experiments. c–e NF-κB, MLK3, MKK3/6, p38, and JNK signaling was measured using antibodies against phospho-NF-κB, phospho-MLK3, phospho-MKK3/6, phospho-p38, phospho-JNK, and β-actin. The maximum phosphoprotein intensity in visfatin-treated samples was set to 100%, and the relative intensities of test samples were calculated. The maximum control phosphoprotein intensity was set to 100%, and relative test intensities were calculated. Data are means ± standard errors of three independent experiments. *p
    Figure Legend Snippet: Effects of signaling inhibitors on visfatin-induced CCL20 expression and release. THP-1 cells were pre-incubated with 5 µM BAY11-7082 (NF-κB inhibitor), 1 µM URMC-099 (MLK3 inhibitor), 10 µM SB 203,580 (p38 MAPK inhibitor), and 10 µM SP 600,125 (JNK inhibitor) for 4 h, and stimulated with visfatin (vis) for 20 h. a Expression of CCL20 was analyzed by real-time PCR. b CCL20 level in culture supernatants were measured by ELISA. Data are means ± standard errors of three independent experiments. c–e NF-κB, MLK3, MKK3/6, p38, and JNK signaling was measured using antibodies against phospho-NF-κB, phospho-MLK3, phospho-MKK3/6, phospho-p38, phospho-JNK, and β-actin. The maximum phosphoprotein intensity in visfatin-treated samples was set to 100%, and the relative intensities of test samples were calculated. The maximum control phosphoprotein intensity was set to 100%, and relative test intensities were calculated. Data are means ± standard errors of three independent experiments. *p

    Techniques Used: Expressing, Incubation, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    27) Product Images from "The New Synthetic H2S-Releasing SDSS Protects MC3T3-E1 Osteoblasts against H2O2-Induced Apoptosis by Suppressing Oxidative Stress, Inhibiting MAPKs, and Activating the PI3K/Akt Pathway"

    Article Title: The New Synthetic H2S-Releasing SDSS Protects MC3T3-E1 Osteoblasts against H2O2-Induced Apoptosis by Suppressing Oxidative Stress, Inhibiting MAPKs, and Activating the PI3K/Akt Pathway

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2017.00007

    Involvement of mitogen-activated protein kinases and the phosphatidylinositol 3-kinase/Akt pathway in the protective effect of SDSS. Representative Western blots and group data showing the effect of SDSS and DSS on H 2 O 2 -stimulated extracellular signal-regulated kinase 1/2 (Erk1/2; A ), p38 (B) , and c-Jun N-terminal kinase (Jnk; C ) phosphorylation. (D) Representative Western blots and group data showing that SDSS (25 and 100 μM) induced Akt phosphorylation. (E,F) LY294002 (15 μM, 15 min) attenuated the effect of SDSS (25 μM, 18 h) on cell viability (E) , and messenger RNA (mRNA) expression levels of heme oxygenase 1 (HO-1) and glutamate-cysteine ligase modifier subunit (GCLM) (F) . Mean ± standard error of the mean, n = 4–6. ∗ p
    Figure Legend Snippet: Involvement of mitogen-activated protein kinases and the phosphatidylinositol 3-kinase/Akt pathway in the protective effect of SDSS. Representative Western blots and group data showing the effect of SDSS and DSS on H 2 O 2 -stimulated extracellular signal-regulated kinase 1/2 (Erk1/2; A ), p38 (B) , and c-Jun N-terminal kinase (Jnk; C ) phosphorylation. (D) Representative Western blots and group data showing that SDSS (25 and 100 μM) induced Akt phosphorylation. (E,F) LY294002 (15 μM, 15 min) attenuated the effect of SDSS (25 μM, 18 h) on cell viability (E) , and messenger RNA (mRNA) expression levels of heme oxygenase 1 (HO-1) and glutamate-cysteine ligase modifier subunit (GCLM) (F) . Mean ± standard error of the mean, n = 4–6. ∗ p

    Techniques Used: Western Blot, Expressing

    28) Product Images from "Kallikrein-related peptidase 6 induces chemotherapeutic resistance by attenuating auranofin-induced cell death through activation of autophagy in gastric cancer"

    Article Title: Kallikrein-related peptidase 6 induces chemotherapeutic resistance by attenuating auranofin-induced cell death through activation of autophagy in gastric cancer

    Journal: Oncotarget

    doi: 10.18632/oncotarget.13352

    Interaction of KLK6, LC3B, and p53 is required for AF-induced autophagy activation A . Western blot analysis of the indicated proteins in cytosolic and nuclear extracts of AGS (top) and NCI-N87 (bottom) cells treated with AF or DMSO for the indicated times. β-actin and PARP served as cytosolic and nuclear loading controls, respectively. B . Colocalization of KLK6 and LC3B on gastric cancer cells. AGS and NCI-N87 cells were treated with AF, and stained at 4°C with KLK6 and LC3B antibody. After primary staining, also stained with Alexa488 (green) and Alexa555 (red), and nuclear stained with DAPI. Colocalization is represented by yellow appearance in the merge. Western blot analysis of KLK6, LC3B, and p53 co-immunoprecipitated (IP) with anti-KLK6 C . anti-LC3B D . and anti-p53 E . from AF-induced chemosensitive (AGS and SNU-216) and chemoresistant (NCI-N87 and SNU-620) cells treated with AF for the indicated times using p53, KLK6, and LC3B antibodies. F . Viability (top) and Western blot analysis of total and phosphorylated Akt and p38 (bottom) in AGS and SNU-216 cells treated with Akt inhibitor VIII (10 μM), p38 inhibitor SB203580 (30 μM), MEK1, 2 inhibitor U0126 (20 μM), and JNK inhibitor II (20 μM). * P
    Figure Legend Snippet: Interaction of KLK6, LC3B, and p53 is required for AF-induced autophagy activation A . Western blot analysis of the indicated proteins in cytosolic and nuclear extracts of AGS (top) and NCI-N87 (bottom) cells treated with AF or DMSO for the indicated times. β-actin and PARP served as cytosolic and nuclear loading controls, respectively. B . Colocalization of KLK6 and LC3B on gastric cancer cells. AGS and NCI-N87 cells were treated with AF, and stained at 4°C with KLK6 and LC3B antibody. After primary staining, also stained with Alexa488 (green) and Alexa555 (red), and nuclear stained with DAPI. Colocalization is represented by yellow appearance in the merge. Western blot analysis of KLK6, LC3B, and p53 co-immunoprecipitated (IP) with anti-KLK6 C . anti-LC3B D . and anti-p53 E . from AF-induced chemosensitive (AGS and SNU-216) and chemoresistant (NCI-N87 and SNU-620) cells treated with AF for the indicated times using p53, KLK6, and LC3B antibodies. F . Viability (top) and Western blot analysis of total and phosphorylated Akt and p38 (bottom) in AGS and SNU-216 cells treated with Akt inhibitor VIII (10 μM), p38 inhibitor SB203580 (30 μM), MEK1, 2 inhibitor U0126 (20 μM), and JNK inhibitor II (20 μM). * P

    Techniques Used: Activation Assay, Western Blot, Staining, Immunoprecipitation

    29) Product Images from "Shikonin inhibits proliferation of melanoma cells by MAPK pathway-mediated induction of apoptosis"

    Article Title: Shikonin inhibits proliferation of melanoma cells by MAPK pathway-mediated induction of apoptosis

    Journal: Bioscience Reports

    doi: 10.1042/BSR20203834

    Western blot analysis of MAPK pathway in melanoma cells ( A ) A375SM cells were treated with indicated concentrations of shikonin for 24 h and harvested to measure expression levels of MAPK pathways; ERK, JNK, and p38 proteins were detected by Western blot analysis. ( B ) Bar graph was generated by quantifying blots from three independent experiments using ImageJ and normalizing the intensity of the bands to the DMSO-treated control. Significance was determined by Student’s t test, * P
    Figure Legend Snippet: Western blot analysis of MAPK pathway in melanoma cells ( A ) A375SM cells were treated with indicated concentrations of shikonin for 24 h and harvested to measure expression levels of MAPK pathways; ERK, JNK, and p38 proteins were detected by Western blot analysis. ( B ) Bar graph was generated by quantifying blots from three independent experiments using ImageJ and normalizing the intensity of the bands to the DMSO-treated control. Significance was determined by Student’s t test, * P

    Techniques Used: Western Blot, Expressing, Generated

    Shikonin induced apoptosis in melanoma tumor tissues ( A ) Apoptosis was measured in tumor tissues using the TUNEL assay and ( C ) phospho-p38 expression was measured in tumor tissues by immunohistochemistry. ( B ) TUNEL-positive and ( D ) p-p38-positive cells were observed under a light microscope and are shown as the average of four fields. Significance was determined by Student’s t test, * P
    Figure Legend Snippet: Shikonin induced apoptosis in melanoma tumor tissues ( A ) Apoptosis was measured in tumor tissues using the TUNEL assay and ( C ) phospho-p38 expression was measured in tumor tissues by immunohistochemistry. ( B ) TUNEL-positive and ( D ) p-p38-positive cells were observed under a light microscope and are shown as the average of four fields. Significance was determined by Student’s t test, * P

    Techniques Used: TUNEL Assay, Expressing, Immunohistochemistry, Light Microscopy

    30) Product Images from "MAPK Phosphatase-1 Deficiency Exacerbates the Severity of Imiquimod-Induced Psoriasiform Skin Disease"

    Article Title: MAPK Phosphatase-1 Deficiency Exacerbates the Severity of Imiquimod-Induced Psoriasiform Skin Disease

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.00569

    MAPK phosphatase-1 (MKP-1) negatively regulates cytokine expression through inhibition of p38 activity in macrophages. (A–C) Bone marrow (BM) cells from wild-type (WT) and MKP-1 −/− mice were used for in vitro dendritic cell culture, neutrophil isolation, and macrophage culture. Il1b mRNA expression was examined in BM-derived DCs (A) , neutrophils (B) , and BM-derived macrophages (C) after being treated with R848 or LPS for 5 h. (D) The abundances of phosphorylated IκBα, ERK, and p38 were analyzed by Western blot (left), and the relative expression was normalized by GAPDH (right). (E) WT and MKP-1 −/− macrophages were treated with R848 for 5 h in the presence or absence of SB203580 to examine the expression of Il1b, Cxcl2 , and S100a8 . Data are representative of two independent experiments.
    Figure Legend Snippet: MAPK phosphatase-1 (MKP-1) negatively regulates cytokine expression through inhibition of p38 activity in macrophages. (A–C) Bone marrow (BM) cells from wild-type (WT) and MKP-1 −/− mice were used for in vitro dendritic cell culture, neutrophil isolation, and macrophage culture. Il1b mRNA expression was examined in BM-derived DCs (A) , neutrophils (B) , and BM-derived macrophages (C) after being treated with R848 or LPS for 5 h. (D) The abundances of phosphorylated IκBα, ERK, and p38 were analyzed by Western blot (left), and the relative expression was normalized by GAPDH (right). (E) WT and MKP-1 −/− macrophages were treated with R848 for 5 h in the presence or absence of SB203580 to examine the expression of Il1b, Cxcl2 , and S100a8 . Data are representative of two independent experiments.

    Techniques Used: Expressing, Inhibition, Activity Assay, Mouse Assay, In Vitro, Cell Culture, Isolation, Derivative Assay, Western Blot

    Inhibition of p38 activity alleviates the disease severity of MKP-1 −/− mice. Wild-type (WT) and MKP-1 −/− mice were topically treated with imiquimod cream for five consecutive days and received p38 inhibitor SB203580 or DMSO (the solvent of Sb203580) via intraperitoneally injection daily. n = 4–5 mice per group. (A) Changes in ear thickness. (B) Representative images of hematoxylin and eosin staining in skin section (scale bars: 50 µm). Data are representative of two independent experiments.
    Figure Legend Snippet: Inhibition of p38 activity alleviates the disease severity of MKP-1 −/− mice. Wild-type (WT) and MKP-1 −/− mice were topically treated with imiquimod cream for five consecutive days and received p38 inhibitor SB203580 or DMSO (the solvent of Sb203580) via intraperitoneally injection daily. n = 4–5 mice per group. (A) Changes in ear thickness. (B) Representative images of hematoxylin and eosin staining in skin section (scale bars: 50 µm). Data are representative of two independent experiments.

    Techniques Used: Inhibition, Activity Assay, Mouse Assay, Injection, Staining

    31) Product Images from "Induction of Chemoresistance by All-Trans Retinoic Acid via a Noncanonical Signaling in Multiple Myeloma Cells"

    Article Title: Induction of Chemoresistance by All-Trans Retinoic Acid via a Noncanonical Signaling in Multiple Myeloma Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0085571

    Effect of ATRA on p38-MSK cascade activation in myeloma cells. ( A ) U266 cells were treated with the indicated concentrations of ATRA for 2 h. Cell lysates were subjected to immunoblotting with antibodies to phosphorylated and total p38 as well as MSK1 proteins. ( B ) U266 cells were incubated with 1 µM of ATRA for the indicated periods. Phosphorylated p38 and MSK1 were measured by Western blotting. The experiment was repeated at least three times. The figure shows a representative result.
    Figure Legend Snippet: Effect of ATRA on p38-MSK cascade activation in myeloma cells. ( A ) U266 cells were treated with the indicated concentrations of ATRA for 2 h. Cell lysates were subjected to immunoblotting with antibodies to phosphorylated and total p38 as well as MSK1 proteins. ( B ) U266 cells were incubated with 1 µM of ATRA for the indicated periods. Phosphorylated p38 and MSK1 were measured by Western blotting. The experiment was repeated at least three times. The figure shows a representative result.

    Techniques Used: Activation Assay, Incubation, Western Blot

    Essential role of p38-MSK activation in Ape/Ref-1 induction. ( A ) U266 cells were incubated with various signal transduction inhibitors (1 mM NAC, 10 µM SB203580, 10 µM H89) in the presence and absence of 1 µM of ATRA for 48 h. Medium and inhibitors were replaced every 24 h. Ape/Ref-1 and GAPDH proteins were detected by Western blotting and semi-quantitative densitometric analysis was performed from three independent experiments. ( B ) After transfection with β-gal, p38α(CA), and MSK1 plasmids for 48 h, U266 cells were lysed for Western blotting followed by densitometric scanning to quantify the relative expression of Ape/Ref-1 and GAPDH proteins. The experiment was repeated at least three times. The figure shows a representative result. ( C ) U266 cells were transiently transfected with expression constructs for β-gal, p38α(KD), and MSK1(CKD). At 24 h after transfection, cells were incubated with 1 µM ATRA for another 48 h. Cell lysates were assayed for Ape/Ref-1 and GAPDH expression. All results represent at least three independent experiments. ( D ) U266 cells were transfected with 100 nM of control or MSK1 siRNA for 24 h, and then incubated with 1 µM ATRA for another 48 h. Ape/Ref-1 and GAPDH levels were measured and shown in representative blots. The relative levels of Ape/Ref-1 to GAPDH in each group from A was quantified by densitometric analysis and normalized to that of the control group. Data are mean ± SD from three independent experiments. * P
    Figure Legend Snippet: Essential role of p38-MSK activation in Ape/Ref-1 induction. ( A ) U266 cells were incubated with various signal transduction inhibitors (1 mM NAC, 10 µM SB203580, 10 µM H89) in the presence and absence of 1 µM of ATRA for 48 h. Medium and inhibitors were replaced every 24 h. Ape/Ref-1 and GAPDH proteins were detected by Western blotting and semi-quantitative densitometric analysis was performed from three independent experiments. ( B ) After transfection with β-gal, p38α(CA), and MSK1 plasmids for 48 h, U266 cells were lysed for Western blotting followed by densitometric scanning to quantify the relative expression of Ape/Ref-1 and GAPDH proteins. The experiment was repeated at least three times. The figure shows a representative result. ( C ) U266 cells were transiently transfected with expression constructs for β-gal, p38α(KD), and MSK1(CKD). At 24 h after transfection, cells were incubated with 1 µM ATRA for another 48 h. Cell lysates were assayed for Ape/Ref-1 and GAPDH expression. All results represent at least three independent experiments. ( D ) U266 cells were transfected with 100 nM of control or MSK1 siRNA for 24 h, and then incubated with 1 µM ATRA for another 48 h. Ape/Ref-1 and GAPDH levels were measured and shown in representative blots. The relative levels of Ape/Ref-1 to GAPDH in each group from A was quantified by densitometric analysis and normalized to that of the control group. Data are mean ± SD from three independent experiments. * P

    Techniques Used: Activation Assay, Incubation, Transduction, Western Blot, Transfection, Expressing, Construct

    32) Product Images from "Aqueous Extract of Gracilaria tenuistipitata Suppresses LPS-Induced NF-?B and MAPK Activation in RAW 264.7 and Rat Peritoneal Macrophages and Exerts Hepatoprotective Effects on Carbon Tetrachloride-Treated Rat"

    Article Title: Aqueous Extract of Gracilaria tenuistipitata Suppresses LPS-Induced NF-?B and MAPK Activation in RAW 264.7 and Rat Peritoneal Macrophages and Exerts Hepatoprotective Effects on Carbon Tetrachloride-Treated Rat

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0086557

    Effect of AEGT on MAP kinase phosphorylation in LPS-stimulated RAW 264.7 cells. The RAW264.7 cells were seeded in a 24-well plate, treated with 400 or 800 µg/ml AEGT for 1 h and stimulated with 1 µg/ml LPS for 24 h. The cell lysates were collected, and p38, JNK, and ERK1/2 phosphorylation was analyzed by western blot analysis with specific antibodies. The relative value of the LPS- group or AEGT/LPS-treated group over the untreated group was measured by densitometry following normalization to cellular GAPDH levels. The basal level of promoter activity in the AEGT/LPS-untreated cells was defined as 1. Error bars indicate the means ± SD of three independent experiments. * P
    Figure Legend Snippet: Effect of AEGT on MAP kinase phosphorylation in LPS-stimulated RAW 264.7 cells. The RAW264.7 cells were seeded in a 24-well plate, treated with 400 or 800 µg/ml AEGT for 1 h and stimulated with 1 µg/ml LPS for 24 h. The cell lysates were collected, and p38, JNK, and ERK1/2 phosphorylation was analyzed by western blot analysis with specific antibodies. The relative value of the LPS- group or AEGT/LPS-treated group over the untreated group was measured by densitometry following normalization to cellular GAPDH levels. The basal level of promoter activity in the AEGT/LPS-untreated cells was defined as 1. Error bars indicate the means ± SD of three independent experiments. * P

    Techniques Used: Western Blot, Activity Assay

    33) Product Images from "Checkpoint Kinase ATR Phosphorylates Cdt2, a Substrate Receptor of CRL4 Ubiquitin Ligase, and Promotes the Degradation of Cdt1 following UV Irradiation"

    Article Title: Checkpoint Kinase ATR Phosphorylates Cdt2, a Substrate Receptor of CRL4 Ubiquitin Ligase, and Promotes the Degradation of Cdt1 following UV Irradiation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0046480

    Cdt2 phosphorylation after UV irradiation is caffeine- and MAP kinase-inhibitor sensitive. A. HeLa cells synchronized in the G1 phase were treated with the indicated inhibitors for 2 h and UV-irradiated (+) or not (−). Cell extract was blotted with Cdt1 and Cdt2 antibodies. Inhibitors used were: ERKi, U0126; JNKi, SP600125; p38i, SB202190. The asterisk indicates a non-specific band, and used as a loading control. B. Asynchronously growing HeLa cells were treated with the indicated MAP kinase inhibitors and UV-irradiated (+) or not. One hour later, cells were examined for Cdt2 phosphorylation levels and Cdt1 protein levels. Cdt2 phosphorylation levels were examined using Phos-tag gel. C. Target specificity of inhibitors. Activation of each MAP kinase was examined using specific phosphopeptide antibodies (Erk1 and 2, JNK or p38) before (−) and after (+) UV-irradiation (C-1 and 2). Cells, treated or not with the indicated inhibitors for 2 h, were UV-irradiated or not. One hour later, cell extracts were prepared and their phosphorylation levels of ERK (Erk1 and Erk2), Jun, and MAPKAPK2 (MK2) were examined(C-2). Note that U0126 inhibits the ERK pathway by inhibiting ERK1-activating kinases MEK1 and MEK2. Jun is a JNK substrate and MAPKAPK2 is a p38 substrate. Arrow in MK2 Western blotting indicates the phosphorylated form of MK2. The asterisk indicates a non-specific band.
    Figure Legend Snippet: Cdt2 phosphorylation after UV irradiation is caffeine- and MAP kinase-inhibitor sensitive. A. HeLa cells synchronized in the G1 phase were treated with the indicated inhibitors for 2 h and UV-irradiated (+) or not (−). Cell extract was blotted with Cdt1 and Cdt2 antibodies. Inhibitors used were: ERKi, U0126; JNKi, SP600125; p38i, SB202190. The asterisk indicates a non-specific band, and used as a loading control. B. Asynchronously growing HeLa cells were treated with the indicated MAP kinase inhibitors and UV-irradiated (+) or not. One hour later, cells were examined for Cdt2 phosphorylation levels and Cdt1 protein levels. Cdt2 phosphorylation levels were examined using Phos-tag gel. C. Target specificity of inhibitors. Activation of each MAP kinase was examined using specific phosphopeptide antibodies (Erk1 and 2, JNK or p38) before (−) and after (+) UV-irradiation (C-1 and 2). Cells, treated or not with the indicated inhibitors for 2 h, were UV-irradiated or not. One hour later, cell extracts were prepared and their phosphorylation levels of ERK (Erk1 and Erk2), Jun, and MAPKAPK2 (MK2) were examined(C-2). Note that U0126 inhibits the ERK pathway by inhibiting ERK1-activating kinases MEK1 and MEK2. Jun is a JNK substrate and MAPKAPK2 is a p38 substrate. Arrow in MK2 Western blotting indicates the phosphorylated form of MK2. The asterisk indicates a non-specific band.

    Techniques Used: Irradiation, Activation Assay, Western Blot

    34) Product Images from "Anthrax lethal toxin rapidly reduces c-Jun levels by inhibiting c-Jun gene transcription and promoting c-Jun protein degradation"

    Article Title: Anthrax lethal toxin rapidly reduces c-Jun levels by inhibiting c-Jun gene transcription and promoting c-Jun protein degradation

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M117.805648

    Anthrax LT treatment reduces c-Jun by disrupting MAPK signaling pathways. A and B , Hepa1c1c7 cells were left untreated ( M ) or treated with LT for 1, 2, and 4 h. Cell lysates were subjected to Western blot analysis for assessing protein levels of various MKKs ( A ) and total and phosphorylated Erk1/2, p38, and JNK1/2 ( B ). C , Hepa1c1c7 cells were left untreated or treated with the MAPK inhibitors U0126, SB203580, and SP600125 for 1, 2, 3, and 4 h. The levels of c-Jun protein in treated cells were determined by Western blotting. β-Actin was used as a loading control. Data shown are representative of three independent experiments. The intensities of protein bands were quantified using the Image Studio software of the Odyssey system and normalized by the amounts of β-actin; relative amounts are shown below each lane.
    Figure Legend Snippet: Anthrax LT treatment reduces c-Jun by disrupting MAPK signaling pathways. A and B , Hepa1c1c7 cells were left untreated ( M ) or treated with LT for 1, 2, and 4 h. Cell lysates were subjected to Western blot analysis for assessing protein levels of various MKKs ( A ) and total and phosphorylated Erk1/2, p38, and JNK1/2 ( B ). C , Hepa1c1c7 cells were left untreated or treated with the MAPK inhibitors U0126, SB203580, and SP600125 for 1, 2, 3, and 4 h. The levels of c-Jun protein in treated cells were determined by Western blotting. β-Actin was used as a loading control. Data shown are representative of three independent experiments. The intensities of protein bands were quantified using the Image Studio software of the Odyssey system and normalized by the amounts of β-actin; relative amounts are shown below each lane.

    Techniques Used: Western Blot, Software

    35) Product Images from "Dichloroacetate potentiates tamoxifen-induced cell death in breast cancer cells via downregulation of the epidermal growth factor receptor"

    Article Title: Dichloroacetate potentiates tamoxifen-induced cell death in breast cancer cells via downregulation of the epidermal growth factor receptor

    Journal: Oncotarget

    doi: 10.18632/oncotarget.10999

    p38 MAPK-mediated EGFR degradation following combined treatment of DCA with tamoxifen ( A ) MCF7 cells were treated with 10 μM tamoxifen and 20 mM DCA for 24 h and then treated with or without 50 μg/ml cycloheximide (CHX) for the indicated times. ( B ) MCF7 cells were treated with 10 μM tamoxifen and 20 mM DCA for 24 h and then treated with or without 5 mM MG132 for 24 h. ( C ) MCF7 cells were treated with 10 μM tamoxifen and 20 mM DCA for the indicated times. ( D ) MCF7 cells were pretreated with the indicated doses of SB203580 for 1 h and then treated with 10 μM tamoxifen and 20 mM DCA for 48 h. Protein extracts were harvested and examined by Western blotting. Representative results from independent experiments conducted in triplicate with similar results are shown.
    Figure Legend Snippet: p38 MAPK-mediated EGFR degradation following combined treatment of DCA with tamoxifen ( A ) MCF7 cells were treated with 10 μM tamoxifen and 20 mM DCA for 24 h and then treated with or without 50 μg/ml cycloheximide (CHX) for the indicated times. ( B ) MCF7 cells were treated with 10 μM tamoxifen and 20 mM DCA for 24 h and then treated with or without 5 mM MG132 for 24 h. ( C ) MCF7 cells were treated with 10 μM tamoxifen and 20 mM DCA for the indicated times. ( D ) MCF7 cells were pretreated with the indicated doses of SB203580 for 1 h and then treated with 10 μM tamoxifen and 20 mM DCA for 48 h. Protein extracts were harvested and examined by Western blotting. Representative results from independent experiments conducted in triplicate with similar results are shown.

    Techniques Used: Western Blot

    36) Product Images from "Activation of AMPKα2 in adipocytes is essential for nicotine-induced insulin resistance in vivo"

    Article Title: Activation of AMPKα2 in adipocytes is essential for nicotine-induced insulin resistance in vivo

    Journal: Nature medicine

    doi: 10.1038/nm.3826

    Nicotine treatment results in greater pMKP1-Ser334 levels and subsequent degradation through AMPK. ( a ) MKP1 expression (top) and its serine (Ser) /threonine (Thr) phosphorylation (bottom) in WAT from Veh- or Nic-treated mice. Gapdh was detected as a loading control; n = 8 each. ( b ) AMPKα2-MKP1binding in MEF-derived adipocytes; n = 5 each. ( c ) AMPK activation and MKP1 serine phosphorylation in nicotine-treated isolated adipocytes; n = 4 each. ( d ) MKP1 phosphorylation in HEK293 cells transfected with vector, GFP-tagged WT MKP1 (WT) or the indicated site-directed mutants of MKP1; n = 4 each. ( e ) The indicated peptides (the SAMS peptide and its mutant, SAMSmu) or peptides spanning the indicated amino acid residues of MKP1; n = 3 each. ( f ) MKP1 ubiquitination in HEK293 cells transfected with WT MKP1 (WT) or MKP1-S334A; n = 4 each. ( g ) MKP1 protein expression in MG132-treated isolated adipocytes; n = 5 each. ( h ) The subcellular localization of AMPKα1/2, p38 and MKP1 in adipocytes treated with or without nicotine. LDH (lactate dehydrogenase), cytoplasmic marker; H3 (histone 3), nuclear marker; n = 5 each. ( i ) Insulin signaling (western blot, top) and lipolysis (bottom) in Nic- or Veh-treated MEF-derived adipocytes transfected with adenovirus GFP (Ad-GFP) or MKP1 (Ad-MKP1).; n = 5 each. ( j ) Detection of pMKP1-S334 in WAT from the indicated strains in Nic- or Veh-treated mice (top) and its quantitation (bottom). Beta-actin was detected as a loading control; n = 6 each. Significance determined by one-way ANOVA with Bonferroni’s post-hoc test ( a,i,j ) and * P
    Figure Legend Snippet: Nicotine treatment results in greater pMKP1-Ser334 levels and subsequent degradation through AMPK. ( a ) MKP1 expression (top) and its serine (Ser) /threonine (Thr) phosphorylation (bottom) in WAT from Veh- or Nic-treated mice. Gapdh was detected as a loading control; n = 8 each. ( b ) AMPKα2-MKP1binding in MEF-derived adipocytes; n = 5 each. ( c ) AMPK activation and MKP1 serine phosphorylation in nicotine-treated isolated adipocytes; n = 4 each. ( d ) MKP1 phosphorylation in HEK293 cells transfected with vector, GFP-tagged WT MKP1 (WT) or the indicated site-directed mutants of MKP1; n = 4 each. ( e ) The indicated peptides (the SAMS peptide and its mutant, SAMSmu) or peptides spanning the indicated amino acid residues of MKP1; n = 3 each. ( f ) MKP1 ubiquitination in HEK293 cells transfected with WT MKP1 (WT) or MKP1-S334A; n = 4 each. ( g ) MKP1 protein expression in MG132-treated isolated adipocytes; n = 5 each. ( h ) The subcellular localization of AMPKα1/2, p38 and MKP1 in adipocytes treated with or without nicotine. LDH (lactate dehydrogenase), cytoplasmic marker; H3 (histone 3), nuclear marker; n = 5 each. ( i ) Insulin signaling (western blot, top) and lipolysis (bottom) in Nic- or Veh-treated MEF-derived adipocytes transfected with adenovirus GFP (Ad-GFP) or MKP1 (Ad-MKP1).; n = 5 each. ( j ) Detection of pMKP1-S334 in WAT from the indicated strains in Nic- or Veh-treated mice (top) and its quantitation (bottom). Beta-actin was detected as a loading control; n = 6 each. Significance determined by one-way ANOVA with Bonferroni’s post-hoc test ( a,i,j ) and * P

    Techniques Used: Expressing, Mouse Assay, Derivative Assay, Activation Assay, Isolation, Transfection, Plasmid Preparation, Mutagenesis, Marker, Western Blot, Quantitation Assay

    Adipose AMPKα2 is required for the nicotine-dependent inhibitory effects on weight gain and insulin signaling in mice. Nic or Veh was perfused in control (Con, black), Prkaa1 Δad (brown) and Prkaa2 Δad (purple) mice. ( a ) Phosphorylated AMPK at Thr172 (pAMPK) and phosphorylated ACC (pACC) expression in WAT; n = 8 each. ( b ) Genotypes of adipose-specific knockout mice and the expressions of AMPKα, AMPKα1, and AMPKα2 in isolated adipocytes; n = 8 each. ( c ) Body weight in mice perfused with Veh or Nic; n = 8–9 each. ( d ) Representative H E-stained WAT sections ( n = 10 section each) and the diameters of adipocytes in WAT (Scale bar, 50 µm); n = 8 each. ( e ) Serum FFA levels after overnight fasting (Fasted) or insulin perfusion at 40 min during ITT; n = 8 each. ( f ) IPGTT in mice treated with Veh or Nic; n = 8–9 each. ( g ) Irs1, pIrs1-Ser307 (pIrs1), pAkt-Ser473 (pAkt), p38, and JNK signaling in WAT of mice injected with saline or insulin (1 unit kg −1 ); n = 8 each. Significance determined by Student’s t -test ( a,d ), one-way ANOVA with repeated measures for the inter-assay evaluations ( c,f ), one-way ANOVA with Bonferroni’s post-hoc test ( e,g ) and * P
    Figure Legend Snippet: Adipose AMPKα2 is required for the nicotine-dependent inhibitory effects on weight gain and insulin signaling in mice. Nic or Veh was perfused in control (Con, black), Prkaa1 Δad (brown) and Prkaa2 Δad (purple) mice. ( a ) Phosphorylated AMPK at Thr172 (pAMPK) and phosphorylated ACC (pACC) expression in WAT; n = 8 each. ( b ) Genotypes of adipose-specific knockout mice and the expressions of AMPKα, AMPKα1, and AMPKα2 in isolated adipocytes; n = 8 each. ( c ) Body weight in mice perfused with Veh or Nic; n = 8–9 each. ( d ) Representative H E-stained WAT sections ( n = 10 section each) and the diameters of adipocytes in WAT (Scale bar, 50 µm); n = 8 each. ( e ) Serum FFA levels after overnight fasting (Fasted) or insulin perfusion at 40 min during ITT; n = 8 each. ( f ) IPGTT in mice treated with Veh or Nic; n = 8–9 each. ( g ) Irs1, pIrs1-Ser307 (pIrs1), pAkt-Ser473 (pAkt), p38, and JNK signaling in WAT of mice injected with saline or insulin (1 unit kg −1 ); n = 8 each. Significance determined by Student’s t -test ( a,d ), one-way ANOVA with repeated measures for the inter-assay evaluations ( c,f ), one-way ANOVA with Bonferroni’s post-hoc test ( e,g ) and * P

    Techniques Used: Mouse Assay, Expressing, Knock-Out, Isolation, Staining, Injection, Inter Assay

    MKP1 reduction is required for nicotine-mediated IR and lipolysis. ( a–g ) WT and Mkp1 −/− mice were treated with Veh or Nic. ( a ) Body weight changes and fat mass in mice treated with Veh or Nic; n = 7–8 each. ( b ) In vivo lipolysis. Plasma glycerol concentration measured at 0, 15, 60, 120 min; n = 6 each. ( c,d ) IPGTT ( c ) and ITT ( d ) data in mice; n = 7–8 each. ( e,f ) The glucose infusion rate (GIR) ( e ) and the hepatic glucose production (HGP), the rate of the disappearance (R d ) and in tibialis anterior and soleus muscles or eWAT the glucose metabolic index (R g ) ( f ) during a hyperinsulinemic-euglycemic clamp ( n = 6 each). ( g ) MKP1, pIrs1- Ser307, Irs1 and p38 signaling in WAT of mice of the indicated genotype; n = 6 each. ( h ) AchRα7 expression in isolated adipocytes, hepatocytes, β-cells, and myocytes after treatment with Nic or Veh; n = 6 each. ( i ) Detection of pAMPK, AMPK, MKP1, and Irs1 expression in WAT in smokers and nonsmokers; n = 12 each. ( j ) Oral glucose tolerance test (OGTT) (left), the insulin levels during OGTT (right) and the areas under the curve (AUC); n = 12 each. Significance determined by one-way ANOVA with Bonferroni’s post-hoc test ( a,f,g,j ), one-way ANOVA with repeated measures for the inter-assay evaluations ( b,c,d,e ), Student’s t -test ( h,i ), * P
    Figure Legend Snippet: MKP1 reduction is required for nicotine-mediated IR and lipolysis. ( a–g ) WT and Mkp1 −/− mice were treated with Veh or Nic. ( a ) Body weight changes and fat mass in mice treated with Veh or Nic; n = 7–8 each. ( b ) In vivo lipolysis. Plasma glycerol concentration measured at 0, 15, 60, 120 min; n = 6 each. ( c,d ) IPGTT ( c ) and ITT ( d ) data in mice; n = 7–8 each. ( e,f ) The glucose infusion rate (GIR) ( e ) and the hepatic glucose production (HGP), the rate of the disappearance (R d ) and in tibialis anterior and soleus muscles or eWAT the glucose metabolic index (R g ) ( f ) during a hyperinsulinemic-euglycemic clamp ( n = 6 each). ( g ) MKP1, pIrs1- Ser307, Irs1 and p38 signaling in WAT of mice of the indicated genotype; n = 6 each. ( h ) AchRα7 expression in isolated adipocytes, hepatocytes, β-cells, and myocytes after treatment with Nic or Veh; n = 6 each. ( i ) Detection of pAMPK, AMPK, MKP1, and Irs1 expression in WAT in smokers and nonsmokers; n = 12 each. ( j ) Oral glucose tolerance test (OGTT) (left), the insulin levels during OGTT (right) and the areas under the curve (AUC); n = 12 each. Significance determined by one-way ANOVA with Bonferroni’s post-hoc test ( a,f,g,j ), one-way ANOVA with repeated measures for the inter-assay evaluations ( b,c,d,e ), Student’s t -test ( h,i ), * P

    Techniques Used: Mouse Assay, In Vivo, Concentration Assay, Expressing, Isolation, Inter Assay

    37) Product Images from "Bz-423 Superoxide Signals Apoptosis via Selective Activation of JNK, Bak, and Bax"

    Article Title: Bz-423 Superoxide Signals Apoptosis via Selective Activation of JNK, Bak, and Bax

    Journal: Free radical biology & medicine

    doi: 10.1016/j.freeradbiomed.2008.07.022

    Bz-423 activates JNK. (A) Lysates prepared from MEFs treated with Bz-423 (10 μM) were immunoblotted to detect total and phosphorylated JNK. (B) Following treatment (2 h) with the indicated concentrations of Bz-423 (in μM), total cellular lysates were immunoblotted for total and phospho-p38. (C) Lysates prepared from MEFs treated with Bz-423 (10 μM) were immunoblotted to detect total and phosphorylated c-Jun and ATF2. (D) MEF were pre-treated with MnTBAP (100 μM), vitamin E (100 μM), or vehicle for 30 min prior to treatment with Bz-423 (10 μM, 2 h). Whole cell lysates were immunoblotted for JNK and phospho-JNK expression. The black vertical line indicates where non-adjacent lanes from the same gel were used to compile this figure. (E) Following pre-treatment with SP600125 (10 μM, dashed line) or vehicle (solid line), MEFs were incubated with Bz-423 and viability (24 h) was determined by PI exclusion. P
    Figure Legend Snippet: Bz-423 activates JNK. (A) Lysates prepared from MEFs treated with Bz-423 (10 μM) were immunoblotted to detect total and phosphorylated JNK. (B) Following treatment (2 h) with the indicated concentrations of Bz-423 (in μM), total cellular lysates were immunoblotted for total and phospho-p38. (C) Lysates prepared from MEFs treated with Bz-423 (10 μM) were immunoblotted to detect total and phosphorylated c-Jun and ATF2. (D) MEF were pre-treated with MnTBAP (100 μM), vitamin E (100 μM), or vehicle for 30 min prior to treatment with Bz-423 (10 μM, 2 h). Whole cell lysates were immunoblotted for JNK and phospho-JNK expression. The black vertical line indicates where non-adjacent lanes from the same gel were used to compile this figure. (E) Following pre-treatment with SP600125 (10 μM, dashed line) or vehicle (solid line), MEFs were incubated with Bz-423 and viability (24 h) was determined by PI exclusion. P

    Techniques Used: Expressing, Incubation

    38) Product Images from "Activation of Human Dendritic Cells by Ascophyllan Purified from Ascophyllum nodosum"

    Article Title: Activation of Human Dendritic Cells by Ascophyllan Purified from Ascophyllum nodosum

    Journal: Marine Drugs

    doi: 10.3390/md17010066

    Phosphorylation of mitogen-activated protein kinase (MAPK) signaling pathway in MDDCs by ascophyllan. ( A ) Phosphorylation of ERK, p38 and JNK was analyzed in MDDCs by western blotting after 1 h of ascophyllan (50 μg/mL) or fucoidan (50 μg/mL) treatment. (B and C) MDDCs were pre-treated with PD98059 (ERK inhibitor; 10 μM), SB203580 (p38 inhibitor; 2μM) or SP600125 (JNK inhibitor; 10 μM) for 1 h and then stimulated with ascophyllan for 24 h. ( B ) MFI levels of CD80 and ( C ) CD86 are shown. All data are representative of or the average of analyses of 6 independent samples. ** p
    Figure Legend Snippet: Phosphorylation of mitogen-activated protein kinase (MAPK) signaling pathway in MDDCs by ascophyllan. ( A ) Phosphorylation of ERK, p38 and JNK was analyzed in MDDCs by western blotting after 1 h of ascophyllan (50 μg/mL) or fucoidan (50 μg/mL) treatment. (B and C) MDDCs were pre-treated with PD98059 (ERK inhibitor; 10 μM), SB203580 (p38 inhibitor; 2μM) or SP600125 (JNK inhibitor; 10 μM) for 1 h and then stimulated with ascophyllan for 24 h. ( B ) MFI levels of CD80 and ( C ) CD86 are shown. All data are representative of or the average of analyses of 6 independent samples. ** p

    Techniques Used: Western Blot

    39) Product Images from "Sesamol Induces Human Hepatocellular Carcinoma Cells Apoptosis by Impairing Mitochondrial Function and Suppressing Autophagy"

    Article Title: Sesamol Induces Human Hepatocellular Carcinoma Cells Apoptosis by Impairing Mitochondrial Function and Suppressing Autophagy

    Journal: Scientific Reports

    doi: 10.1038/srep45728

    Effects of sesamol on mitochondrial membrane potential and redox-sensitive signaling in HepG2 cells. Cells were treated with sesamol at the indicated concentrations for 4 or 24 h. After treatment, ( A ) the cells were detected by a multimode reader after staining with 5 μg/mL JC-1, and were photographed by fluorescence microscopy; the bar graph is the fluorescence intensity which was measured using a multimode microplate reader at 485 nm excitation, 585 nm (red/orange for normal MMP) and 538 nm (green for loss of MMP) emission, respectively. (200 × , magnification). ( B ) H 2 O 2 production was determined by the Amplex Red Hydrogen Peroxide/Peroxidase Assay, and ( C ) Left panel is the representative western blots of expressions of redox-sensitive signaling Akt, and MAPK signaling JNK and p38; and the right panel is the representative western blots of expressions of mitochondria complex I subunit ND1 and mitochondrial biogenesis related protein PGC1α, pAMPK/AMPK. Data presented as mean ± SD, n ≥ 6 wells per group, *p
    Figure Legend Snippet: Effects of sesamol on mitochondrial membrane potential and redox-sensitive signaling in HepG2 cells. Cells were treated with sesamol at the indicated concentrations for 4 or 24 h. After treatment, ( A ) the cells were detected by a multimode reader after staining with 5 μg/mL JC-1, and were photographed by fluorescence microscopy; the bar graph is the fluorescence intensity which was measured using a multimode microplate reader at 485 nm excitation, 585 nm (red/orange for normal MMP) and 538 nm (green for loss of MMP) emission, respectively. (200 × , magnification). ( B ) H 2 O 2 production was determined by the Amplex Red Hydrogen Peroxide/Peroxidase Assay, and ( C ) Left panel is the representative western blots of expressions of redox-sensitive signaling Akt, and MAPK signaling JNK and p38; and the right panel is the representative western blots of expressions of mitochondria complex I subunit ND1 and mitochondrial biogenesis related protein PGC1α, pAMPK/AMPK. Data presented as mean ± SD, n ≥ 6 wells per group, *p

    Techniques Used: Staining, Fluorescence, Microscopy, Western Blot

    40) Product Images from "Downregulation of Mitogen-Activated Protein Kinases by the Bordetella bronchiseptica Type III Secretion System Leads to Attenuated Nonclassical Macrophage Activation "

    Article Title: Downregulation of Mitogen-Activated Protein Kinases by the Bordetella bronchiseptica Type III Secretion System Leads to Attenuated Nonclassical Macrophage Activation

    Journal: Infection and Immunity

    doi: 10.1128/IAI.73.1.308-316.2005

    Wild-type B. bronchiseptica transiently activates ERK1/2 phosphorylation in BMDC. The effects of different stimulants (described for Fig. ) on phosphorylation of p38 and ERK1/2 in BMDC are shown. Gates were set on live and CD11c high cells.
    Figure Legend Snippet: Wild-type B. bronchiseptica transiently activates ERK1/2 phosphorylation in BMDC. The effects of different stimulants (described for Fig. ) on phosphorylation of p38 and ERK1/2 in BMDC are shown. Gates were set on live and CD11c high cells.

    Techniques Used:

    Wild-type B. bronchiseptica downregulates p38 and ERK1/2 phosphorylation in BMMΦ and this downregulation precedes cell death. BMMΦ were left untreated or stimulated with 25 μg of LPS per ml for the indicated times (C, D, G, and
    Figure Legend Snippet: Wild-type B. bronchiseptica downregulates p38 and ERK1/2 phosphorylation in BMMΦ and this downregulation precedes cell death. BMMΦ were left untreated or stimulated with 25 μg of LPS per ml for the indicated times (C, D, G, and

    Techniques Used:

    Wild-type B. bronchiseptica downregulates p38 and ERK1/2 phosphorylation in RAW 264.7 cells and this downregulation precedes cell death. Flow cytometric analysis was performed to select live cells that were intracellularly stained for phosphorylated p38
    Figure Legend Snippet: Wild-type B. bronchiseptica downregulates p38 and ERK1/2 phosphorylation in RAW 264.7 cells and this downregulation precedes cell death. Flow cytometric analysis was performed to select live cells that were intracellularly stained for phosphorylated p38

    Techniques Used: Flow Cytometry, Staining

    Wild-type B. bronchiseptica downregulates p38 and ERK 1 and 2 phosphorylation in RAW 264.7 cells after initial type III secretion independent activation. Macrophages were untreated (lane 1), stimulated with 10 μM anisomycin for 15 min (lane 2),
    Figure Legend Snippet: Wild-type B. bronchiseptica downregulates p38 and ERK 1 and 2 phosphorylation in RAW 264.7 cells after initial type III secretion independent activation. Macrophages were untreated (lane 1), stimulated with 10 μM anisomycin for 15 min (lane 2),

    Techniques Used: Activation Assay

    Wild-type B. bronchiseptica downregulates p38 and ERK1/2 kinase activity in RAW 264.7 cells. In vitro kinase assays were performed with immobilized p38 and ERK1/2 (from incubations with cell lysates) and their substrates, ATF-2 and ELK, respectively.
    Figure Legend Snippet: Wild-type B. bronchiseptica downregulates p38 and ERK1/2 kinase activity in RAW 264.7 cells. In vitro kinase assays were performed with immobilized p38 and ERK1/2 (from incubations with cell lysates) and their substrates, ATF-2 and ELK, respectively.

    Techniques Used: Activity Assay, In Vitro

    Wild-type B. bronchiseptica downregulates p38 and ERK1/2 phosphorylation in BMMΦ. Effects of different treatments on the phosphorylation of p38 and ERK1/2 in BMMΦ are shown. BMMΦ were left untreated or stimulated with 25 μg
    Figure Legend Snippet: Wild-type B. bronchiseptica downregulates p38 and ERK1/2 phosphorylation in BMMΦ. Effects of different treatments on the phosphorylation of p38 and ERK1/2 in BMMΦ are shown. BMMΦ were left untreated or stimulated with 25 μg

    Techniques Used:

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    Cell Signaling Technology Inc p38
    Effects of pilloin on MAPK signalling pathways in LPS-stimulated RAW 264.7 macrophages. RAW 264.7 cells (10 6 cells in MP-6 plates) were treated with vehicle, pilloin or the indicated inhibitor for 6 h before incubation with LPS (100 ng/mL) for 20 min. Cell lysates were analysed by Western blot to detect the activation of ( A ) JNK, ( B ) ERK and ( C ) <t>p38.</t> PD98059 (ERK inhibitor), SB203580 (p38 inhibitor) and SP600125 (JNK inhibitor) served as positive controls. * indicates a significant difference versus the LPS-treated group ( p
    P38, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc p p38
    Schema of proposed cold plasma-induced regulation of p53. The primary event in the described pathways is the recognition of plasma-generated reactive oxygen species (ROS) by specific ROS sensors in keratinocytes ( e.g. , transcription factors p53 and Nrf2 and kinases ATM or Keap1). Plasma generates ROS which in turn activate and phosphorylate p53 via upstream kinases. Activation of p53 increases transcription of p53 targets (BAX, CDKN1A, and GADD45), which increases p53-dependent apoptosis and cell death. Increased expression and phosphorylation of heat shock protein HSP27 by <t>p38</t> MAP kinase result in p53 binding. HSP27 protects HaCaT cells from plasma-induced apoptosis by increased transcription of p21 resulting in cell cycle arrest, DNA repair, and cell survival. Plasma-induced activation and phosphorylation of MAP kinases ( e.g. , signal transduction and transcription control) modulates the expression of genes and proteins related to proliferation and cell survival via Erk1/2. Therefore, p53 acts as an anti- and prooxidant.
    P P38, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Effects of pilloin on MAPK signalling pathways in LPS-stimulated RAW 264.7 macrophages. RAW 264.7 cells (10 6 cells in MP-6 plates) were treated with vehicle, pilloin or the indicated inhibitor for 6 h before incubation with LPS (100 ng/mL) for 20 min. Cell lysates were analysed by Western blot to detect the activation of ( A ) JNK, ( B ) ERK and ( C ) p38. PD98059 (ERK inhibitor), SB203580 (p38 inhibitor) and SP600125 (JNK inhibitor) served as positive controls. * indicates a significant difference versus the LPS-treated group ( p

    Journal: Molecules

    Article Title: Pilloin, A Flavonoid Isolated from Aquilaria sinensis, Exhibits Anti-Inflammatory Activity In Vitro and In Vivo

    doi: 10.3390/molecules23123177

    Figure Lengend Snippet: Effects of pilloin on MAPK signalling pathways in LPS-stimulated RAW 264.7 macrophages. RAW 264.7 cells (10 6 cells in MP-6 plates) were treated with vehicle, pilloin or the indicated inhibitor for 6 h before incubation with LPS (100 ng/mL) for 20 min. Cell lysates were analysed by Western blot to detect the activation of ( A ) JNK, ( B ) ERK and ( C ) p38. PD98059 (ERK inhibitor), SB203580 (p38 inhibitor) and SP600125 (JNK inhibitor) served as positive controls. * indicates a significant difference versus the LPS-treated group ( p

    Article Snippet: Antibodies against phospho-IkB-α (Ser32), IkB-α, COX-2, Erk, phospho-Erk1/2 (Thr202/Tyr204), JNK, phospho-JNK (Thr183/Tyr185), p38, and phospho-p38 (Thr180/Tyr182) were from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Incubation, Western Blot, Activation Assay

    The inhibitory effects of NC-8 on the RANKL-induced osteoclast differentiation signaling pathway. ( A and B ) RAW 264.7 cells were treated with 100 ng/ml RANKL or with 10 μg/ml NC-8, and the protein was collected after the reaction. Western blot was used to detect MAPK pathway and downstream NFATc1, c-Fos and NF-κB protein molecules. ( A ) Relative protein expressions analysis of proteins related to the MAPK signaling pathway, including p-ERK, p-JNK, and p-p38. β-actin was used as a loading control. ( B ) Relative protein expressions analysis of proteins related to the NF-κB signaling pathway, including p65, NFATc1 and c-Fos. β-actin was used as a loading control. Relative expression levels of p65, NFATc1 and c-Fos protein in the cytoplasm and in the nuclear fractions. β-actin and HDAC1 were used as loading controls. The results are expressed as the mean ± S.E.M. of four independent experiments. *p

    Journal: Scientific Reports

    Article Title: Isosteviol Derivative Inhibits Osteoclast Differentiation and Ameliorates Ovariectomy-Induced Osteoporosis

    doi: 10.1038/s41598-018-29257-1

    Figure Lengend Snippet: The inhibitory effects of NC-8 on the RANKL-induced osteoclast differentiation signaling pathway. ( A and B ) RAW 264.7 cells were treated with 100 ng/ml RANKL or with 10 μg/ml NC-8, and the protein was collected after the reaction. Western blot was used to detect MAPK pathway and downstream NFATc1, c-Fos and NF-κB protein molecules. ( A ) Relative protein expressions analysis of proteins related to the MAPK signaling pathway, including p-ERK, p-JNK, and p-p38. β-actin was used as a loading control. ( B ) Relative protein expressions analysis of proteins related to the NF-κB signaling pathway, including p65, NFATc1 and c-Fos. β-actin was used as a loading control. Relative expression levels of p65, NFATc1 and c-Fos protein in the cytoplasm and in the nuclear fractions. β-actin and HDAC1 were used as loading controls. The results are expressed as the mean ± S.E.M. of four independent experiments. *p

    Article Snippet: The blots were blocked with 5% non-fat dry milk in Tris-buffered saline containing 0.5% Tween-20 (TBST) for 1 h at room temperature and were then probed with rabbit anti-mouse antibodies against NF-κB, p-p38, p-ERK, p-JNK, p38, ERK, JNK, c-Fos, and NFATc1 (1:1000; all from Cell Signaling) for 1 h at room temperature.

    Techniques: Western Blot, Expressing

    Schema of proposed cold plasma-induced regulation of p53. The primary event in the described pathways is the recognition of plasma-generated reactive oxygen species (ROS) by specific ROS sensors in keratinocytes ( e.g. , transcription factors p53 and Nrf2 and kinases ATM or Keap1). Plasma generates ROS which in turn activate and phosphorylate p53 via upstream kinases. Activation of p53 increases transcription of p53 targets (BAX, CDKN1A, and GADD45), which increases p53-dependent apoptosis and cell death. Increased expression and phosphorylation of heat shock protein HSP27 by p38 MAP kinase result in p53 binding. HSP27 protects HaCaT cells from plasma-induced apoptosis by increased transcription of p21 resulting in cell cycle arrest, DNA repair, and cell survival. Plasma-induced activation and phosphorylation of MAP kinases ( e.g. , signal transduction and transcription control) modulates the expression of genes and proteins related to proliferation and cell survival via Erk1/2. Therefore, p53 acts as an anti- and prooxidant.

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Cold Physical Plasma Modulates p53 and Mitogen-Activated Protein Kinase Signaling in Keratinocytes

    doi: 10.1155/2019/7017363

    Figure Lengend Snippet: Schema of proposed cold plasma-induced regulation of p53. The primary event in the described pathways is the recognition of plasma-generated reactive oxygen species (ROS) by specific ROS sensors in keratinocytes ( e.g. , transcription factors p53 and Nrf2 and kinases ATM or Keap1). Plasma generates ROS which in turn activate and phosphorylate p53 via upstream kinases. Activation of p53 increases transcription of p53 targets (BAX, CDKN1A, and GADD45), which increases p53-dependent apoptosis and cell death. Increased expression and phosphorylation of heat shock protein HSP27 by p38 MAP kinase result in p53 binding. HSP27 protects HaCaT cells from plasma-induced apoptosis by increased transcription of p21 resulting in cell cycle arrest, DNA repair, and cell survival. Plasma-induced activation and phosphorylation of MAP kinases ( e.g. , signal transduction and transcription control) modulates the expression of genes and proteins related to proliferation and cell survival via Erk1/2. Therefore, p53 acts as an anti- and prooxidant.

    Article Snippet: After blocking in TBS-T (0.05% nonfat milk powder in TRIS-buffered saline pH 7.6/0.05% Tween 20,TBS-T), blots were incubated with Erk1/2 (#9102), Mek1/2 (#9126), Sapk/Jnk (#9258), p38 (#9212), p53 (#2527) as well as phospho-specific antibodies for p-ATM (S1981, #5883), p-ATR (S428, #2853), p-Chk1 (S296, #2349), p-Chk2 (T68, #2661), p-Erk1/2 (T202/Y204, #4370), p-p38 (T180/Y182, #9216), p-Mek1/2 (S217/S221, #9154), p-Sapk/Jnk (T183/Y185, #4668), p-HSP27 (S78,# 2405), p-p53 (S15, # 9286), and p-p53 (S37, #2989), all 1 : 1000 in TBS-T at 4°C overnight (Cell Signaling Technologies, Germany).

    Techniques: Generated, Activation Assay, Expressing, Binding Assay, Transduction

    Plasma-induced activation of MAP kinase signaling in HaCaT keratinocytes. Displayed are relative phosphorylation levels of Erk (p-T202/Y204, a), Jnk (p-T183/Y185 (b)), and p38 (p-T180/Y182, (c) kinases after different treatment times. Lower panels showed the results for time course of relative phosphorylation levels after 180 s of plasma treatment for Erk (d), Jnk (e), and p38 (f). Each expression was normalized to total protein expression. Representative blots are shown. Data are presented as mean + S.D. of two analyses. The x -axis represents treatment time (a–c) or incubation after plasma treatment (d–f). Statistical analysis was done using one-way ANOVA with Dunnett corrections for multiple comparisons to untreated, normalized control ( ∗ p

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Cold Physical Plasma Modulates p53 and Mitogen-Activated Protein Kinase Signaling in Keratinocytes

    doi: 10.1155/2019/7017363

    Figure Lengend Snippet: Plasma-induced activation of MAP kinase signaling in HaCaT keratinocytes. Displayed are relative phosphorylation levels of Erk (p-T202/Y204, a), Jnk (p-T183/Y185 (b)), and p38 (p-T180/Y182, (c) kinases after different treatment times. Lower panels showed the results for time course of relative phosphorylation levels after 180 s of plasma treatment for Erk (d), Jnk (e), and p38 (f). Each expression was normalized to total protein expression. Representative blots are shown. Data are presented as mean + S.D. of two analyses. The x -axis represents treatment time (a–c) or incubation after plasma treatment (d–f). Statistical analysis was done using one-way ANOVA with Dunnett corrections for multiple comparisons to untreated, normalized control ( ∗ p

    Article Snippet: After blocking in TBS-T (0.05% nonfat milk powder in TRIS-buffered saline pH 7.6/0.05% Tween 20,TBS-T), blots were incubated with Erk1/2 (#9102), Mek1/2 (#9126), Sapk/Jnk (#9258), p38 (#9212), p53 (#2527) as well as phospho-specific antibodies for p-ATM (S1981, #5883), p-ATR (S428, #2853), p-Chk1 (S296, #2349), p-Chk2 (T68, #2661), p-Erk1/2 (T202/Y204, #4370), p-p38 (T180/Y182, #9216), p-Mek1/2 (S217/S221, #9154), p-Sapk/Jnk (T183/Y185, #4668), p-HSP27 (S78,# 2405), p-p53 (S15, # 9286), and p-p53 (S37, #2989), all 1 : 1000 in TBS-T at 4°C overnight (Cell Signaling Technologies, Germany).

    Techniques: Activation Assay, Expressing, Incubation

    IL-10 promotes the proliferation of AML cells. (A) IL-10 levels were increased in plasma and bone marrow plasma in 20 AML patients compared with 10 healthy donors. Unpaired t -test was used to determine the differences. (B) The CD4 + CD25 high ICOS + cells and CD4 + CD25 high ICOS − cells were sorted and cultured in 200 μl X-VIVO™ 15 supplemented with 20 ng/ml IL-2 for 2 days, and the supernatants were subsequently collected and the levels of IL-10 were determined using a commercial ELISA kits. Data were expressed as mean ± SEM representing four independent experiments and unpaired t -test was used to determine the difference. (C) Treatment with IL-10 for 24 h promoted the proliferation of HL-60 cells and HEL cells in a dose-dependent manner. Data were expressed as mean ± SEM representing five independent experiments and ANOVA was used to determine the differences. (D) IL-10 promoted the phosphorylation of Akt, Erk1/2, p38, and Stat3. Images shown were representatives of three independent experiments.

    Journal: Frontiers in Immunology

    Article Title: Acute Myeloid Leukemia Cells Express ICOS Ligand to Promote the Expansion of Regulatory T Cells

    doi: 10.3389/fimmu.2018.02227

    Figure Lengend Snippet: IL-10 promotes the proliferation of AML cells. (A) IL-10 levels were increased in plasma and bone marrow plasma in 20 AML patients compared with 10 healthy donors. Unpaired t -test was used to determine the differences. (B) The CD4 + CD25 high ICOS + cells and CD4 + CD25 high ICOS − cells were sorted and cultured in 200 μl X-VIVO™ 15 supplemented with 20 ng/ml IL-2 for 2 days, and the supernatants were subsequently collected and the levels of IL-10 were determined using a commercial ELISA kits. Data were expressed as mean ± SEM representing four independent experiments and unpaired t -test was used to determine the difference. (C) Treatment with IL-10 for 24 h promoted the proliferation of HL-60 cells and HEL cells in a dose-dependent manner. Data were expressed as mean ± SEM representing five independent experiments and ANOVA was used to determine the differences. (D) IL-10 promoted the phosphorylation of Akt, Erk1/2, p38, and Stat3. Images shown were representatives of three independent experiments.

    Article Snippet: The protein was boiled and subjected to western blot with antibodies against Akt, p-Akt (Ser473), Erk1/2, p-Erk1/2 (Thr202/Tyr204), p38, p-p38 (Thr180/Tyr182), Stat3, p-Stat3 (Tyr705) or GAPDH (all from Cell Signaling Technology, Beverly, MA, USA), respectively.

    Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay