Journal: Genes & Development

Article Title: tRNA synthetase activity is required for stress granule and P-body assembly

doi: 10.1101/gad.353535.125

Figure Lengend Snippet: tRNA synthetase inhibition causes persistent ribosome stalls. ( A ) U-2 OS cells expressing GFP-G3BP1 were treated with 20 µM halofuginone (HF) or 0.2% DMSO carrier. Images were collected every 2 h for 16 h and representative images are shown ( n = 3 independent experiments). ( B ) Cells were treated with DMSO (−) or 20 µM HF for 4 or 16 h, and P-eIF2α and total eIF2α were detected by Western blotting. Representative blots with total protein are shown at the left , and the average ± SEM of n = 3 independent experiments is shown at the right . Molecular weights (in kilodaltons) are shown. ( C ) Cells were treated with 20 µM HF for 1 h (black line), 4 h (purple line), or 16 h (green line), and polysome profiling was performed. A representative profile is shown at the left , and the average ± SEM of the polysome:monosome ratio from n = 3 independent replicates is shown at the right . Pink, gray, and green dots represent results from each replicate. ( D ) Cells were treated with either 0.2% DMSO carrier or 20 µM HF in the presence or absence of 250 µM sodium arsenite (As) or 2 µM rocaglamide A (RocA), and GFP-G3BP1 was imaged every 0.5 h for 3 h. Representative images are shown at the left with the average ± SEM. The percentage of cells with SGs from n = 3 independent experiments is shown at the right ; ≥288 cells were counted per treatment across all replicates. ( E ) Cells were treated with 0.2% DMSO (black line), 2 µM RocA (green line), or 2 µM RocA plus 20 µM HF (purple line) for 3 h followed by polysome profiling. Representative profiles are shown at the left , and the average ± SEM from n = 3 independent experiments is shown at the right , with pink, gray, and green dots representing each replicate. ( F ) Cells were untreated (DMSO carrier) or treated for 24, 4, or 1 h with 20 µM anisomycin (ANI) or 20 µM halofuginone, and Western blotting for p-p38 and p-JNK was done, with the total protein shown below . A representative blot of n = 2 independent experiments is shown. Scale bars, 10 µm. Significance was assessed with ordinary one-way ANOVAs and Tukey's multiple comparisons tests. (*) P < 0.05, (**) P < 0.01, (***) P < 0.005.

Article Snippet: p-p38 (Thr180/Tyr182) , Proteintech , 28796 , 1:1000 (Western).

Techniques: Inhibition, Expressing, Western Blot

Journal: Cancer Research

Article Title: Dysfunctional Microvasculature as a Consequence of Shb Gene Inactivation Causes Impaired Tumor Growth

doi: 10.1158/0008-5472.can-08-3797

Figure Lengend Snippet: Figure 5. Cytoskeleton and VEGF-dependent signaling in cultured endothelial cells isolated from Shb knockout or wild-type liver. A, the cytoskeleton of isolated endothelial cells maintained in tissue culture for 5 to 7 d was visualized by staining with rhodamine-phalloidin, showing a less regular shape with numerous extensions in Shb null cells. B, corresponding wild-type control. No clear cytoskeletal difference between wild-type or knockout cells was noted after stimulation with VEGF-A (C, Shb knockout; D, Shb wild type). Addition of VEGF to the control cells (D) produced changes that made the cells resemble the Shb null cells in the absence of VEGF-A. Horizontal scale bar is shown. Equal amounts of protein from the experimental groups (+/, 20 ng/mL VEGF-A for 2 min) were subjected to Western blot analysis for the phosphorylated proteins indicated. Shb blot shows unstimulated cells only. The blots were subjected to densitometric analysis for phosphorylated FAK, total FAK, phosphorylated p38, total p38, pMLC, phosphorylated ERK, and total ERK in three separate experiments. Quantitation of the relative phosphorylation of FAK, MLC, p38, and ERK is also given. Columns, mean; bars, SE. *, P < 0.05; **, P < 0.01 (paired Students’ t test).

Article Snippet: The samples were electrophoresed on SDS-polyacrylamide gels, protein was transferred on to Hybond-P filters (GE Healthcare), and these were then probed for pY-1175 VEGFR-2, total VEGFR-2, pY-397 FAK, total FAK, phosphorylated extracellular signal-regulated kinase (ERK), total ERK, and phosphorylated Akt, total Akt, phosphorylated myosin light chain (pMLC), pY-658 VE-cadherin, phosphorylated p38, and total p38 (all antibodies were from Cell Signaling, except for total VEGFR-2 from R&D Systems, pY-397 FAK from Biosource, pY-VE-cadherin from ProSci, and pMLC from Santa Cruz Biotechnology) before incubation with secondary antibodies and enhanced chemiluminescence.

Techniques: Cell Culture, Isolation, Knock-Out, Staining, Control, Produced, Western Blot, Quantitation Assay, Phospho-proteomics

Journal: Molecular and cellular endocrinology

Article Title: Estrogen receptor beta maintains expression of KLF15 to prevent cardiac myocyte hypertrophy in female rodents

doi: 10.1016/j.mce.2017.11.004

Figure Lengend Snippet: (a) TGFβ stimulates p38α activity, inhibited by SB2036580 or β-LGND. Bar graph is the mean±SD from 3 exps combined. *p < 0.05 vs. control, +p < 0.05 for TGFβ vs same + SB2036580 or β-LGND. (b) TGFβ inhibits KLF15 mRNA and protein in cardiomyocytes, blocked by the p38 antagonist SB2036580 (0.1μM) (c) TAK1 activating phosphorylation is stimulated by AngII or TGFβ, inhibited by β-LGND. *p<0.05 vs. control, + p<0.05 for TGFβ or AngII vs same plus β-LGND, n=3 exps. (d) TAK1 siRNA diminishes TGFβ or AngII-stimulated p38α activity. The latter was seen as phosphorylation at tyrosine182. *p<0.05 vs control, +p<0.05 for TGFβ or AngII vs same + β-LGND, n=3 exps. TAK1 siRNA validation is also shown. (e) Flow cytometry analysis of β-LGND inhibition of phospho-kinases due to cAMP/PKA. *p<0.05 for control vs. AngII-stimulated phospho-TAK1, phospho-p38α, or KLF15 proteins. +p<0.05 for AngII vs AngII + β-LGND, ++p<0.05 for AngII + β-LGND vs same + either H-89 (PKA inhibitor) or RP-8-Br-cAMP (cAMP inhibitor), n=3 exps.

Article Snippet: Additional antibodies and phospho-specific antibodies used for immuno-blots were obtained from the followings: Cell Signaling Technology (Danvers, MA) TAK1 (D94D7) (#5206), Phospho-ATF-2 (Thr71) (#9221), Phospho-TAK1 (Thr187) (#4536); Santa Cruz, Biotechnology (Dallas, TX), KLF15 (A5) (SC-271675), GAPDH (0411) (sc-47724), MYH7 (A4.951) (sc-53090), Actin (2Q1055) (sc-58673), p38 Antibody (A-20) (sc-535), phospho-p38 (Thr 180/Tyr 182) (sc-17852-R); (Boster Biological Technology, Pleasanton, CA), ACTA2 (M01072–1).

Techniques: Activity Assay, Control, Phospho-proteomics, Biomarker Discovery, Flow Cytometry, Inhibition

Journal: Molecular and cellular endocrinology

Article Title: Estrogen receptor beta maintains expression of KLF15 to prevent cardiac myocyte hypertrophy in female rodents

doi: 10.1016/j.mce.2017.11.004

Figure Lengend Snippet: AngII acting through TGFβ stimulates a TAK1-p38α kinase axis that inhibits KLF15 expression and nuclear localization of the protein. This contributes to increased gene expression and cardiomyocyte hypertrophy. ERβ acting through protein kinase A opposes TAK1-p38α activation. This restores KLF15 abundance and nuclear localization, contributing in part to inhibition of AngII-induced gene expression and cardiomyocyte hypertrophy.

Article Snippet: Additional antibodies and phospho-specific antibodies used for immuno-blots were obtained from the followings: Cell Signaling Technology (Danvers, MA) TAK1 (D94D7) (#5206), Phospho-ATF-2 (Thr71) (#9221), Phospho-TAK1 (Thr187) (#4536); Santa Cruz, Biotechnology (Dallas, TX), KLF15 (A5) (SC-271675), GAPDH (0411) (sc-47724), MYH7 (A4.951) (sc-53090), Actin (2Q1055) (sc-58673), p38 Antibody (A-20) (sc-535), phospho-p38 (Thr 180/Tyr 182) (sc-17852-R); (Boster Biological Technology, Pleasanton, CA), ACTA2 (M01072–1).

Techniques: Expressing, Gene Expression, Activation Assay, Inhibition

Journal: Cancer discovery

Article Title: p38MAPK plays a crucial role in stromal mediated tumorigenesis

doi: 10.1158/2159-8290.CD-13-0743

Figure Lengend Snippet: (A) Western blot analysis demonstrating that SB203580 treatment inhibits p38MAPK activity. As expected, SB203580 treatment significantly impacts phosphorylation of p38MAPK’s direct downstream target, Hsp27. Total Hsp27 was used as loading control.

Article Snippet: The primary antibodies used were: polyclonal AUF1 (Millipore, Billerica, MA, catalog number 07260MI) at 1:3000, polyclonal p-p38 (PhosphoSolutions, Aurora, CO, catalog number p190-1802) at 1:1000, polyclonal p38 (Cell Signaling, Boston, MA, catalog number 9218) at 1:1000, monoclonal β-catenin (BD Biosciences, San Jose, CA, catalog number 610153) at 1:5000, and monoclonal α-tubulin (Abcam, Cambridge, MA, product number ab6160) at 1:1000.

Techniques: Western Blot, Activity Assay, Phospho-proteomics, Control

Journal: Cancer discovery

Article Title: p38MAPK plays a crucial role in stromal mediated tumorigenesis

doi: 10.1158/2159-8290.CD-13-0743

Figure Lengend Snippet: (A) RNA-seq analysis was performed on young fibroblasts, senescent fibroblasts, and senescent fibroblasts treated with SB203580. RNA-seq results were analyzed to determine the number of factors upregulated in response to senescence (SASP factors) and the number of p38MAPK-dependent factors. These results were also analyzed for overlap with the expression profiles of breast cancer (BC)-associated stroma.

Article Snippet: The primary antibodies used were: polyclonal AUF1 (Millipore, Billerica, MA, catalog number 07260MI) at 1:3000, polyclonal p-p38 (PhosphoSolutions, Aurora, CO, catalog number p190-1802) at 1:1000, polyclonal p38 (Cell Signaling, Boston, MA, catalog number 9218) at 1:1000, monoclonal β-catenin (BD Biosciences, San Jose, CA, catalog number 610153) at 1:5000, and monoclonal α-tubulin (Abcam, Cambridge, MA, product number ab6160) at 1:1000.

Techniques: RNA Sequencing, Expressing

Journal: Journal of Biological Chemistry

Article Title: JNK and Ceramide Kinase Govern the Biogenesis of Lipid Droplets through Activation of Group IVA Phospholipase A2

doi: 10.1074/jbc.m109.061515

Figure Lengend Snippet: FIGURE 3. FBS induces the phosphorylation of JNK, p38, and ERK in CHO-K1cells.Serum-starvedCHO-K1cellsweretreatedwith7.5%FBSforthe indicated times. Total and phosphorylated JNK, p38, and ERK were monitored by Western blot.

Article Snippet: Rabbit anti-cPLA2 , anti-phospho-Ser-505-cPLA2 , anti-JNK, anti-phospho-Thr-183/Tyr-185-JNK, anti-p38, antiphospho-Thr-180/Tyr-182-p38, anti-p44/42, and anti-phosphoThr-202/Tyr-204-p44/p42 antibodies were from Cell Signaling; chicken anti-ADRP was from GenWay Biotech; rabbit anti-glyceraldehyde-3-phosphate dehydrogenase was from Ambion, and rabbit anti-CERK fromAbcam.

Techniques: Phospho-proteomics, Western Blot

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Hibiscus sabdariffa L.: A potent natural neuroprotective agent for the prevention of streptozotocin-induced Alzheimer's disease in mice.

doi: 10.1016/j.biopha.2020.110303

Figure Lengend Snippet: Fig. 6. Effects of Hibiscus extracts and celecoxib on the activity of p38MAPK (A), and COX-2 (B), and on concentration of mPGES-1 (C) and PGE2 (D). p38MAPK and COX-2 levels in celecoxib (30 mg/kg), white and red Hibiscus (200 mg/kg each) groups were decreased compared to STZ (3 mg/kg, ICV) group and increased compared to the normal group. mPGES-1 and PGE2levels were normalized in the three treated groups. Panel 4 represents immunoblot analysis shown in Fig. 6.A. Panel 5 represents immunoblot analysis shown in Fig. 6.B. Data are expressed as means ± S.D. The significance of the dif- ference between means was tested by ANOVA followed by Tukey Kramer multiple compar- isons test. * P < 0.05 vs normal; @ P < 0.05 vs STZ-treated group; # P < 0.05 vs celecoxib. n = 6 mice, DF = 29. For p38MAPK: F = 354.1, R2 = 0.9827. For COX-2: F = 350.5, R2 = 0.9825. For m-PGES-1: F = 430.4, R2 = 0.9857. For PGE2: F = 354.3, R2 = 0.9827.

Article Snippet: The antibodies used were rabbit BACE1 monoclonal [EPR19523] (abcam, ab183612), mouse Cox-2 (H-3) monoclonal (Santa cruze Inc, sc-376861), rabbit PSENEN polyclonal (Boster Biological Technology, A04504) and rabbit p38 MAPK polyclonal (Biorbyt Ltd., orb127559).

Techniques: Activity Assay, Concentration Assay, Western Blot