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: 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: Molecular Biology of the Cell

Article Title: Localization and retention of p90 ribosomal S6 kinase 1 in the nucleus: implications for its function

doi: 10.1091/mbc.E11-07-0658

Figure Lengend Snippet: RSK1 phosphorylation by PDK1 is required for its nuclear translocation. (A) Schematic of phosphorylation sites on rat RSK1. (B) Top two rows, serum-starved HeLa cells were treated with and without EGF (100 nM) for 10 min and the localization of total RSK1 was monitored with anti-RSK1 antibody; 4′,6-diamidino-2-phenylindole staining shows nuclei. Bottom four rows, the PDK1 inhibitor BX795 inhibits nuclear translocation of RSK1. HeLa cells were treated as described above with and without overnight preincubation with 0.5 μM BX795. The localization of active RSK1 was monitored with anti–phospho-T573-RSK antibody. (C) BX795 inhibits phosphorylation of RSK1 on S221 and S732. Cells were treated as in B. After immunoprecipitation with anti-RSK1 antibody, Western analyses of the immune complexes were performed with anti–phospho RSK antibodies. Scale bar, 10 μm. Representative data from three independent experiments are shown.

Article Snippet: Anti–phospho-RSK1/2 (S221) from R&D Systems (Minneapolis, MN), anti–phospho-RSK (T359/S363) from Cell Signaling (Beverly, MA), anti–phospho-RSK (S380) from Epitomics (Burlingame, CA), anti–phospho-RSK (T573) from Cell Signaling, and anti–phospho-RSK S732 raised by Rockland (Gilbertsville, PA) were used.

Techniques: Phospho-proteomics, Translocation Assay, Staining, Immunoprecipitation, Western Blot

Journal: Neurochemical Research

Article Title: Exogenous TIPE2 Inhibit TAK1 to Improve Inflammation and Neuropathic Pain Induced by Sciatic Nerve Injury Through Inactivating NF-κB and JNK

doi: 10.1007/s11064-022-03671-4

Figure Lengend Snippet: TIPE2 inhibited activation of NF-κB and JNK via downregulation of TAK1. A Immunofluorescence staining was performed to analyze phosphor-JNK in spinal cord and DRG after 14 days. Mean fluorescence intensity of phospho-JNK was analyzed by Image J 1.49p. The scale bar was 20 μm. B Western blot was carried out to analyze the expression of p-NF-κB p65, p-JNK1, and p-JNK2 in spinal cord after 14 days. && p < 0.01 versus Sham; % p < 0.05, %% p < 0.01 versus Model; $ p < 0.05, $$ p < 0.01 versus TIPE2-1; ! p < 0.05, !! p < 0.01 versus TIPE2-5

Article Snippet: After blocking by 5% non-fat milk, the membranes were incubated with primary antibodies against p-IκBα (1:10000, ab133462, Abcam), IκBα (1:800, ab95338, Abcam), p-JNK1(1:1200, ab47337, Abcam), p-JNK2 (1:1000, P02706, Boster), JNK1 (1:2500, ab199380, Abcam), JNK2 (1:1500, 51153-1-AP, Proteintech), β-actin (1:2000, 20536-1-AP, Proteintech), TIPE2 (1:1000, orb158628, Biorbyt), TAK1 (1:800, orb256677, Biorbyt) at 4 °C overnight.

Techniques: Activation Assay, Immunofluorescence, Staining, Fluorescence, Western Blot, Expressing

Journal: BMC Cancer

Article Title: MicroRNA-21 promotes hepatocellular carcinoma HepG2 cell proliferation through repression of mitogen-activated protein kinase-kinase 3

doi: 10.1186/1471-2407-13-469

Figure Lengend Snippet: The expression of MAP2K3 in human HCC tissues determined by IHC

Article Snippet: The membranes were probed with rabbit anti-MAP2K3 antibody and anti-GAPDH antibody (Boster, Wuhan, China) or (1:200, Boster, Wuhan, China) were for the interested protein MAP2K3 and endogenous GAPDH for loading control, respectively.

Techniques: Expressing, Immunohistochemistry

Journal: BMC Cancer

Article Title: MicroRNA-21 promotes hepatocellular carcinoma HepG2 cell proliferation through repression of mitogen-activated protein kinase-kinase 3

doi: 10.1186/1471-2407-13-469

Figure Lengend Snippet: Validation of MAP2K3 mRNA as a target of miR-21. (A) : Sequence of potential binding site of miR-21 in the 3’UTR of MAP2K3 mRNA (top panel), mutations were introduced into the binding site for generation of mutated MAP2K3 3’TUR (bottom panel). ( B and C ): Validation of miR-21 target using MAP2K3 3’UTR luciferase reporter. Cells co-transfected with pMIR-Report/MAP2K3 3’UTR (WT) or pMIR-Report/Mut-MAP2K3 3’UTR (Mut) and pAd/pri-miR-21 (B) , pAd/miR-21/inhibitor (C) , and pAd/con plasmids showed a decreased luciferase activity in pAd/pri-miR-21 cells (B) . Luciferase activity after site directed mutagenesis of the 3’UTR of MAP2K3 mRNA in the miR-21 seed sequence (pMIR-Report/Mut-MAP2K3) was significantly higher with respect to the pMIR-Report/MAP2K3 vector transfected cells ( B and C ). Results represented the mean ± SD from three independent triplicated experiments (N=9).

Article Snippet: The membranes were probed with rabbit anti-MAP2K3 antibody and anti-GAPDH antibody (Boster, Wuhan, China) or (1:200, Boster, Wuhan, China) were for the interested protein MAP2K3 and endogenous GAPDH for loading control, respectively.

Techniques: Biomarker Discovery, Sequencing, Binding Assay, Luciferase, Transfection, Activity Assay, Mutagenesis, Plasmid Preparation

Journal: BMC Cancer

Article Title: MicroRNA-21 promotes hepatocellular carcinoma HepG2 cell proliferation through repression of mitogen-activated protein kinase-kinase 3

doi: 10.1186/1471-2407-13-469

Figure Lengend Snippet: miR-21 targets MAP2K3 mRNA. The HepG2 cells were infected with Ad/pri-miR-21, Ad/miR-21/inhibitor or Ad/con adenoviral vector. The expression of MAP2K3 was detected by immunoblotting analysis against anti-MAP2K3 antibody. Compared with Ad/con group, *: p <0.05. Data in A represented the mean ± SD from three independent triplicated experiments (N=9).

Article Snippet: The membranes were probed with rabbit anti-MAP2K3 antibody and anti-GAPDH antibody (Boster, Wuhan, China) or (1:200, Boster, Wuhan, China) were for the interested protein MAP2K3 and endogenous GAPDH for loading control, respectively.

Techniques: Infection, Plasmid Preparation, Expressing, Western Blot