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: 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