p38 mapk inhibition assay  (MedChemExpress)

Journal: Journal of Experimental Pharmacology

Article Title: Ouabain inhibits monocyte activation in vitro: prevention of the proinflammatory mCD14 + /CD16 + subset appearance and cell-size progression

doi: 10.2147/JEP.S35507

Figure Lengend Snippet: Role of p38MAPK inhibition in the modulation of monocyte cell-size progression induced by ouabain (Oua). Notes: Monocytes were maintained for adhesion in culture for 2 hours and then incubated with 100 nM Oua for 24 hours, in the presence or absence of the p38MAPK inhibitor SB202190 (used at 20 µM). Values refer to the medians of large monocytes in each condition (evaluating only CD14 + cells); a statistical difference from control ( P < 0.01, paired t -test); b statistical difference from Oua + SB202190 ( P < 0.05, paired t -test). Inset: Representative experiment showing monocyte cell-size and cell-granularity profiles obtained by flow cytometry, where gates delimiting small and large subpopulations are depicted, as well as both Oua and SB202190 effects on large-monocyte subpopulation number. These experiments were performed using six individuals.

Article Snippet: The observation that p38 MAPK inhibition influences both ouabain-induced downregulation of mCD14 and ouabain-induced cell size–progression restraint in culture, seen in the present work, raised the idea that p38 MAPK might play a central role in the cell-signaling events triggered by this hormone in monocytes.

Techniques: Inhibition, Incubation, Flow Cytometry

Journal: Journal of Experimental Pharmacology

Article Title: Ouabain inhibits monocyte activation in vitro: prevention of the proinflammatory mCD14 + /CD16 + subset appearance and cell-size progression

doi: 10.2147/JEP.S35507

Figure Lengend Snippet: Effect of the p38 MAPK inhibitor SB202190 on ouabain-induced CD69 upregulation in human monocytes

Article Snippet: The observation that p38 MAPK inhibition influences both ouabain-induced downregulation of mCD14 and ouabain-induced cell size–progression restraint in culture, seen in the present work, raised the idea that p38 MAPK might play a central role in the cell-signaling events triggered by this hormone in monocytes.

Techniques:

Journal: The FASEB Journal

Article Title: Pancreatic cancer‐derived exosomes induce apoptosis of T lymphocytes through the p38 MAPK‐mediated endoplasmic reticulum stress

doi: 10.1096/fj.201902186r

Figure Lengend Snippet: FIGURE 5 Exosomes activate p38 MAPK signaling pathway in T-lymphocyte apoptosis. A, Overlap of core enrichment in apoptosis gene set with genes up-regulated by exosomes and genes involved in MAPK signaling pathway. B, IL1A, IL1B, JUN, and CHOP genes were screened out. The expressions of JUN and CHOP mRNA in Exo-T and Ctrl-T were detected by qPCR. C, Western blotting was performed with anti-JUN antibody and (D) anti-p38, -phospho-p38, -JNK, -phospho-JNK, -ERK, and -phospho-ERK antibody. Blots were reprobed with anti-β-actin antibody to ensure equal loading. Bar diagram indicates relative fold changes normalized to Ctrl-T. Results are expressed as mean ± SD of three independent experiments (N = 3). A value of P < .05 indicates statistically significant differences

Article Snippet: For p38 MAPK inhibition, peripheral T lymphocytes from the other three volunteers were adjusted to a density of 1 × 106/mL, and pre-stimulated with SB203580 (MedChem Express, Monmouth Junction, NJ, USA) for 24 hours before co-incubation with exosomes.

Techniques: Western Blot

Journal: The FASEB Journal

Article Title: Pancreatic cancer‐derived exosomes induce apoptosis of T lymphocytes through the p38 MAPK‐mediated endoplasmic reticulum stress

doi: 10.1096/fj.201902186r

Figure Lengend Snippet: FIGURE 6 Exosome-induced apoptosis depend on phosphorylation of p38 MAPK. A, T lymphocytes were treated with various concentrations of SB203580 for 24 hours. Western blotting was performed with anti-p38 and -phospho-p38 antibodies. Phosphorylation of p38 MAPK was significantly reduced at a concentration of 200 µM. B, T lymphocytes were treated with various concentrations of U0126 for 24 hours. Western blotting was performed with anti-ERK and -phospho-ERK antibodies. Phosphorylation of ERK was significantly reduced at a concentration of 10 µM. C, T lymphocytes were treated with SB203580 (200 μM), exosomes, or combined. Apoptotic rates of Ctrl-T, SB-T, Exo-T, and Exo + SB-T were detected by Annexin V-PI staining and analyzed by flow cytometry. (D) T lymphocytes were treated with U0126 (10 μM), exosomes, or combined. Apoptotic rates of Ctrl-T, U0126-T, Exo-T, and Exo + U0126-T were detected by Annexin V-PI staining and analyzed by flow cytometry. Bar diagram indicates relative fold changes normalized to Ctrl-T. Results are expressed as mean ± SD of three independent experiments (N = 3). A value of P < .05 indicates statistically significant differences. E, Kinetics of p38 phosphorylation in peripheral T cells in response to exosomes. Black slope indicates that the degree of phosphorylation was increased alone with prolonged exosome treatment

Article Snippet: For p38 MAPK inhibition, peripheral T lymphocytes from the other three volunteers were adjusted to a density of 1 × 106/mL, and pre-stimulated with SB203580 (MedChem Express, Monmouth Junction, NJ, USA) for 24 hours before co-incubation with exosomes.

Techniques: Phospho-proteomics, Western Blot, Concentration Assay, Staining, Flow Cytometry

Journal: The FASEB Journal

Article Title: Pancreatic cancer‐derived exosomes induce apoptosis of T lymphocytes through the p38 MAPK‐mediated endoplasmic reticulum stress

doi: 10.1096/fj.201902186r

Figure Lengend Snippet: FIGURE 8 Link between PC-derived exosomes, SB203580, p38MAPK, ER stress, and apoptosis in T lymphocytes. PC-derived exosomes were integrated by T lymphocytes and activated the p38 MAPK in T lymphocytes. Phosphorylated p38 MAPK induced the activation of ER stress. Subsequently, the PERK–eIF2α–ATF4–CHOP signaling axis was activated, and apoptosis was induced. In addition, DUSP1 was also up-regulated by activated p38 MAPK, which was reported to dephosphorylate p38 MAPK (green dotted line), and might serve as a feedback control mechanism for MAPK signaling. Treatment of SB203580 inhibited activated p38 MAPK, reduced ER stress, and protected T lymphocytes from apoptosis. Solid line indicates the result from our study, and dotted line indicates the result from reported research

Article Snippet: For p38 MAPK inhibition, peripheral T lymphocytes from the other three volunteers were adjusted to a density of 1 × 106/mL, and pre-stimulated with SB203580 (MedChem Express, Monmouth Junction, NJ, USA) for 24 hours before co-incubation with exosomes.

Techniques: Derivative Assay, Activation Assay, Control

Journal: The FASEB Journal

Article Title: Pancreatic cancer‐derived exosomes induce apoptosis of T lymphocytes through the p38 MAPK‐mediated endoplasmic reticulum stress

doi: 10.1096/fj.201902186r

Figure Lengend Snippet: FIGURE 7 Role of p38 MAPK in exosome-induced ER stress and apoptosis in T lymphocytes. T lymphocytes were treated with SB203580, exosomes, or combined. A, Western blotting was performed with anti-p38, -phospho-p38, -JUN, -PERK, -eIF2α -phospho-eIF2α, -ATF4, and -CHOP antibodies. Blots were reprobed with anti-β-actin antibody to ensure equal loading. B, Apoptosis is analyzed through Western blotting, which is performed with anti-cleaved-caspase-3 and -cleaved-PARP antibodies. Bar diagram indicates relative fold changes normalized to Ctrl-T. Results are expressed as mean ± SD of three independent experiments (N = 3). A value of P < .05 indicates statistically significant differences. Note: Bar diagram of JUN indicates relative fold changes normalized to Exo-T because its expression in Ctrl-T is too low to detect. C, BxPC-3 (5000/well) was added to each culture well. After BxPC-3 adhered and started to grow (black arrow), Ctrl-T (1 × 105/well), SB203580-treated T cells (SB-T) (1 × 105/well), Exo-T (1 × 105/well), or SB203580-treated Exo-T cells (Exo + SB-T) (1 × 105/well) were added to the co-culture. BxPC-3 that was not co-cultured with T cells was used as a positive internal reference. Ctrl-T and SB-T exhibited stronger antitumor activity; Exo-T showed weaker effectiveness. Compared with Exo-T, the cytotoxic activity of Exo + SB-T was significantly enhanced

Article Snippet: For p38 MAPK inhibition, peripheral T lymphocytes from the other three volunteers were adjusted to a density of 1 × 106/mL, and pre-stimulated with SB203580 (MedChem Express, Monmouth Junction, NJ, USA) for 24 hours before co-incubation with exosomes.

Techniques: Western Blot, Expressing, Co-Culture Assay, Cell Culture, Activity Assay

Journal: Nature structural & molecular biology

Article Title: ADAR1 controls apoptosis of stressed cells by inhibiting Staufen1-mediated mRNA decay.

doi: 10.1038/nsmb.3403

Figure Lengend Snippet: Figure 1 Stress-induced cytoplasmic localization of ADAR1p110. (a) Schematic domain structure of two ADAR1 isoforms. (b) UV-irradiation- induced cytoplasmic localization of mCherry-ADAR1p110-WT in A172 cells, which is blocked by the p38 inhibitor SB203580. (c) Heat-shock- induced cytoplasmic localization of mCherry-ADAR1p110-WT in A172 cells, which is blocked by the p38 inhibitor SB203580. Bar graphs in b and c show quantification from images of at least 180 cells (~30 cells each from ≥7 separate, independently prepared slides) examined for mCherry- ADAR1p110 distribution between the nucleus and cytoplasm. Data are shown as mean ± s.d. ***P < 0.001; NS, not significant by two-tailed Student’s t test. Source data are available in Supplementary Data Set 1. (d) Time-course analysis of mCherry-ADAR1p110-WT localization after UV irradiation. Scale bars in b–d, 20 µm.

Article Snippet: For p38 MAPK inhibition, the treatment of the cell culture with 10 μM of SB203580 (Selleckchem) started before UV irradiation and continued until recovery of cells.

Techniques: Irradiation, Two Tailed Test

Journal: Nature structural & molecular biology

Article Title: ADAR1 controls apoptosis of stressed cells by inhibiting Staufen1-mediated mRNA decay.

doi: 10.1038/nsmb.3403

Figure Lengend Snippet: Figure 2 Cytoplasmic localization of ADAR1p110 is regulated by p38 MAP kinase signaling. (a) The indicated expression constructs for MAP kinase– activating kinase were cotransfected with mCherry-ADAR1p110-WT into A172 cells or mock transfected as a control. Images of ~200 cells (~30 cells each from ≥7 separate, independently prepared slides) were examined for mCherry-ADAR1p110 distribution between the nucleus and cytoplasm. Data are shown as mean ± s.d. ***P < 0.001 by two-tailed Student’s t test. Source data are available in Supplementary Data Set 1. (b) Phosphorylated p38 (pp38) localizes exclusively in the nucleus in UV-irradiated A172 cells. The p38 inhibitor, although it does not affect the nuclear localization of pp38, blocks the cytoplasmic localization of mCherry-ADAR1p110. (c) UV-irradiation-induced cytoplasmic localization of mCherry-ADAR1p110-WT is blocked by simultaneous knockdown of MSK1 and MSK2. Short interfering RNAs (siRNAs) are indicated with the prefix ‘si’. Scale bars in a–c, 20 µm.

Article Snippet: For p38 MAPK inhibition, the treatment of the cell culture with 10 μM of SB203580 (Selleckchem) started before UV irradiation and continued until recovery of cells.

Techniques: Expressing, Construct, Transfection, Control, Two Tailed Test, Irradiation, Knockdown

Journal: Nature structural & molecular biology

Article Title: ADAR1 controls apoptosis of stressed cells by inhibiting Staufen1-mediated mRNA decay.

doi: 10.1038/nsmb.3403

Figure Lengend Snippet: Figure 3 Phosphorylation of ADAR1p110 at five sites. (a) Phos-tag PAGE and western blotting to detect phosphorylated ADAR1p110. A172 cell extracts were prepared after UV irradiation, transient transfection with the MKK6* expression construct in the absence or presence of the p38 inhibitor SB203580, or simultaneous knockdown of MSK1 and MSK2. λPPase indicates treatment with λ-phosphatase before SDS–PAGE. β-Actin, loading control. (b) Phos-tag SDS–PAGE analysis of FLAG-ADAR1p110 recombinant proteins. Cell extracts were prepared from A172 cells transiently transfected with the FLAG-ADAR1p110 expression construct with or without UV irradiation. Uncropped images of a and b are shown in Supplementary Data Set 2. (c) Phosphorylation sites identified by LC-MS/MS are shown. The dsRBD3 region (T725–P809) is highlighted in yellow. Identified phosphorylation sites are in red. Bottom, numbered dashed lines indicate peptides identified by LC-MS/MS. (d) Model of human ADAR1 dsRBD3 and the downstream region in complex with dsRNA. The region of ADAR1 spanning residues 715–799 belongs to the standard dsRBD, whereas the region downstream of this domain, where T808, T811, S814, S823, S825 are located, is disordered. The lysines K777, K778, and K781 coordinating the dsRNA are indicated. (e) Localization of ADAR1p110 phosphorylation-inhibitory (T/S-to-A) and phosphorylation-mimetic (T/S-to-D) mutants. Scale bar, 20 µm.

Article Snippet: For p38 MAPK inhibition, the treatment of the cell culture with 10 μM of SB203580 (Selleckchem) started before UV irradiation and continued until recovery of cells.

Techniques: Phospho-proteomics, Western Blot, Irradiation, Transfection, Expressing, Construct, Knockdown, SDS Page, Control, Recombinant, Liquid Chromatography with Mass Spectroscopy

Journal: The Journal of Physiology

Article Title: Interleukin‐13 affects the epithelial sodium channel in the intestine by coordinated modulation of STAT6 and p38 MAPK activity

doi: 10.1113/JP271156

Figure Lengend Snippet: A, representative Western blot analysis of both phosphorylated and total ERK1/2, JNK, or p38 proteins after IL‐13 exposition. HT‐29/B6‐GR/MR cells were treated with or without DBA and with or without IL‐13 for 96 h, then rested in media lacking FCS and IL‐13 for 3 h and stimulated again with IL‐13 for 5 min (ERK), 15 min (JNK) or 60 min (p38). Then, total cellular protein was extracted and analysed. B, densitometric measurements were obtained from 3–4 blot membranes representing independent experiments. Data given as means ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001. C, determination of ENaC‐dependent Na+ absorption in the presence or absence of ERK1/2 (U0126) or JNK (SP600125) inhibitors. D, determination of ENaC‐dependent Na+ transport with or without the p38 inhibitor SB202190. Data are given as means ± SEM; n = 6–7, **P < 0.01, ***P < 0.001.

Article Snippet: Specific MAPK inhibitors U0126 (which inhibits the p42/44 extracellular signal‐regulated kinase, upstream kinase MEK 1/2) and SP600125 (which inibits JNK MAPK) (both from Cell Signaling Technology, Frankfurt am Main, Germany), and SB202190 (which inhibits p38 MAPK) (Calbiochem, Darmstadt, Germany), were used at the same concentration (10 μ m ).

Techniques: Western Blot

Journal: The Journal of Physiology

Article Title: Interleukin‐13 affects the epithelial sodium channel in the intestine by coordinated modulation of STAT6 and p38 MAPK activity

doi: 10.1113/JP271156

Figure Lengend Snippet: HT‐29/B6‐GR/MR cells were stimulated with or without DBA and with or without IL‐13 in the presence or absence of baricitinib or AS1517499 for 96 h, then rested FCS and IL‐13 free for 3 h and treated again with IL‐13 for 30 min. Then, total cellular protein was extracted, fractionated by SDS‐Page, and immuno‐probed for phospho‐specific ERK1/2, JNK, p38, or STAT6 expression. Human β‐actin served as loading control. A and B, representative Western blot analyses of the indicated proteins in the absence or presence of baricitinib (A) and the corresponding densitometric measurements (B). C and D, representative Western blot analyses of phospho‐specific p38 MAPK in the absence or presence of AS1517499 (C) and the corresponding densitometric measurements (D). Results in B and D are means ± SEM; n = 3, **P < 0.01, ***P < 0.001.

Article Snippet: Specific MAPK inhibitors U0126 (which inhibits the p42/44 extracellular signal‐regulated kinase, upstream kinase MEK 1/2) and SP600125 (which inibits JNK MAPK) (both from Cell Signaling Technology, Frankfurt am Main, Germany), and SB202190 (which inhibits p38 MAPK) (Calbiochem, Darmstadt, Germany), were used at the same concentration (10 μ m ).

Techniques: SDS Page, Expressing, Western Blot

Journal: The Journal of Physiology

Article Title: Interleukin‐13 affects the epithelial sodium channel in the intestine by coordinated modulation of STAT6 and p38 MAPK activity

doi: 10.1113/JP271156

Figure Lengend Snippet: The stimulation with DBA (dexamethasone, butyrate and aldosterone) leads to the up‐regulation of β‐ and γ‐ENaC subunit as well as SGK1 expression. On the one hand this is achieved via activation of the p38 MAPK and on the other hand by transactivation of target genes via activated mineralocorticoid receptor (MR) and glucocorticoid receptor (GR). IL‐13 binds to the IL‐13Rα1/IL‐4Rα type‐I receptor leading to the activation of JAK1/2. Subsequently, STAT6 is phosphorylated and this in turn leads to the inhibition of the DBA‐stimulated activation of p38 MAPK. These signalling events promote the decrease in DBA‐stimulated ENaC activity due to reduction of β‐ and γ‐ENaC and/or SGK1 expression. Technical terms used in this figure are also listed in the abbreviations.

Article Snippet: Specific MAPK inhibitors U0126 (which inhibits the p42/44 extracellular signal‐regulated kinase, upstream kinase MEK 1/2) and SP600125 (which inibits JNK MAPK) (both from Cell Signaling Technology, Frankfurt am Main, Germany), and SB202190 (which inhibits p38 MAPK) (Calbiochem, Darmstadt, Germany), were used at the same concentration (10 μ m ).

Techniques: Expressing, Activation Assay, Inhibition, Activity Assay