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: Frontiers in Bioengineering and Biotechnology

Article Title: Exosomes derived from miR-26a-5p-modified adipose mesenchymal stem cells improve wound healing by targeting MAP2K4

doi: 10.3389/fbioe.2025.1662095

Figure Lengend Snippet: Interaction between miR-26a-5p and MAP2K4 , as well as cell transfection efficiency and characterization of the isolated exosomes. (A) MAP2K4 was the target of miR-26a-5p by dual luciferase reporter gene assay. N = 3. *: P < 0.05. (B) The miR-26a-5p level in the AMSCs with miR-26a-5p overexpression to verify the cell transfection efficiency. N = 3. *: P < 0.05, vs. AMSCs-NC. (C) Particle size distribution of AMSCs-derived exosomes and miR-26a-5p overexpressed AMSCs-derived exosomes determined by Nanosight. (D) The morphology of AMSCs-derived exosomes and miR-26a-5p overexpressed AMSCs-derived exosomes, visualized by transmission electron microscopy. (E) The expression of exosomes-specific markers (CD63, CD81, and HSP70) and negative control protein (calnexin) in exosomes and cells, detected by Western blot. (F) The level of miR-26a-5p in the AMSCs-derived exosomes and miR-26a-5p overexpressed AMSCs-derived exosomes. N = 3. *: P < 0.05, vs. AMSCs-Exo.

Article Snippet: After blocking with 5% skim milk at 37 °C for 2 h, the membranes were incubated the primary antibodies, including anti-CD63 antibody (1: 1,000, Abclonal, Wuhan, China), anti-CD81 antibody (1: 1,000, Abclonal), anti-HSP70 antibody (1: 1,000, Proteintech, Wuhan, China), anti-calnexin antibody (1: 1,000, Proteintech), anti-MAP2K4 antibody (1: 1,000, Proteintech), anti-COL1A1 antibody (1: 1,000, NOVUS, St. Louis, MO, United States), anti-α-SMA antibody (1: 1,000, Cell Signaling Technology, Boston, MA, United States), anti-TNF-α antibody (1: 1,000, Cell Signaling Technology), and anti-GAPDH antibody (1: 10,000, Proteintech).

Techniques: Transfection, Isolation, Luciferase, Reporter Gene Assay, Over Expression, Derivative Assay, Transmission Assay, Electron Microscopy, Expressing, Negative Control, Western Blot

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Exosomes derived from miR-26a-5p-modified adipose mesenchymal stem cells improve wound healing by targeting MAP2K4

doi: 10.3389/fbioe.2025.1662095

Figure Lengend Snippet: The mRNA expression of related genes, including Map2k4 , Col1a1 , Col2a1 , Col3a1 , α-Sma , Tnf-α , Il1β , Il6 , and Cd31 in the different groups measured by RT-qPCR. N = 3. *: P < 0.05, vs. control; # : P < 0.05, vs. model; $ : vs. AMSCs-agomir-Exo.

Article Snippet: After blocking with 5% skim milk at 37 °C for 2 h, the membranes were incubated the primary antibodies, including anti-CD63 antibody (1: 1,000, Abclonal, Wuhan, China), anti-CD81 antibody (1: 1,000, Abclonal), anti-HSP70 antibody (1: 1,000, Proteintech, Wuhan, China), anti-calnexin antibody (1: 1,000, Proteintech), anti-MAP2K4 antibody (1: 1,000, Proteintech), anti-COL1A1 antibody (1: 1,000, NOVUS, St. Louis, MO, United States), anti-α-SMA antibody (1: 1,000, Cell Signaling Technology, Boston, MA, United States), anti-TNF-α antibody (1: 1,000, Cell Signaling Technology), and anti-GAPDH antibody (1: 10,000, Proteintech).

Techniques: Expressing, Quantitative RT-PCR, Control

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Exosomes derived from miR-26a-5p-modified adipose mesenchymal stem cells improve wound healing by targeting MAP2K4

doi: 10.3389/fbioe.2025.1662095

Figure Lengend Snippet: The protein expression of MAP2K4, COL1A1, α-SMA, and TNF-α in the different groups, determined by Western blot. N = 3. *: P < 0.05, vs. control; # : P < 0.05, vs. model; $ : vs. AMSCs-agomir-Exo.

Article Snippet: After blocking with 5% skim milk at 37 °C for 2 h, the membranes were incubated the primary antibodies, including anti-CD63 antibody (1: 1,000, Abclonal, Wuhan, China), anti-CD81 antibody (1: 1,000, Abclonal), anti-HSP70 antibody (1: 1,000, Proteintech, Wuhan, China), anti-calnexin antibody (1: 1,000, Proteintech), anti-MAP2K4 antibody (1: 1,000, Proteintech), anti-COL1A1 antibody (1: 1,000, NOVUS, St. Louis, MO, United States), anti-α-SMA antibody (1: 1,000, Cell Signaling Technology, Boston, MA, United States), anti-TNF-α antibody (1: 1,000, Cell Signaling Technology), and anti-GAPDH antibody (1: 10,000, Proteintech).

Techniques: Expressing, Western Blot, Control

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Exosomes derived from miR-26a-5p-modified adipose mesenchymal stem cells improve wound healing by targeting MAP2K4

doi: 10.3389/fbioe.2025.1662095

Figure Lengend Snippet: Roles and potential mechanisms of MAP2K4 in wound healing in mice. (A) The wound healing process in the skin defect mice treated with si-NC and si-MAP2K4 at days 0, 4, 8, and 12. N = 6. (B) Quantification analysis of wound healing rate in the skin defect mice treated with si-NC and si-MAP2K4 at days 4, 8, and 12. N = 6. *: P < 0.05, vs. si-NC. (C) The mRNA expression of Map2k4 , Col1a1 , α-Sma , and Tnf-α in the skin defect mice treated with si-NC and si-MAP2K4, measured by RT-qPCR. N = 3. *: P < 0.05, vs. si-NC. (D) The level of miR-26a-5p in the skin tissues of different mice. N = 3. *: P < 0.05, vs. si-NC. (E) The protein expression of MAP2K4, COL1A1, α-SMA, and TNF-α in the skin defect mice treated with si-NC and si-MAP2K4, detected by Western blot. N = 3. *: P < 0.05, vs. si-NC.

Article Snippet: After blocking with 5% skim milk at 37 °C for 2 h, the membranes were incubated the primary antibodies, including anti-CD63 antibody (1: 1,000, Abclonal, Wuhan, China), anti-CD81 antibody (1: 1,000, Abclonal), anti-HSP70 antibody (1: 1,000, Proteintech, Wuhan, China), anti-calnexin antibody (1: 1,000, Proteintech), anti-MAP2K4 antibody (1: 1,000, Proteintech), anti-COL1A1 antibody (1: 1,000, NOVUS, St. Louis, MO, United States), anti-α-SMA antibody (1: 1,000, Cell Signaling Technology, Boston, MA, United States), anti-TNF-α antibody (1: 1,000, Cell Signaling Technology), and anti-GAPDH antibody (1: 10,000, Proteintech).

Techniques: Expressing, Quantitative RT-PCR, 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