Journal: eLife

Article Title: Registered report: Diverse somatic mutation patterns and pathway alterations in human cancers

doi: 10.7554/eLife.11566

Figure Lengend Snippet:

Article Snippet: MAP2K4 WT Myc-DDK tagged –includes FLAG tag 1 , Plasmid , Origene , RC206051 , Original product number not specified.

Techniques: Plasmid Preparation, FLAG-tag, Variant Assay, Mutagenesis

Journal: eLife

Article Title: Registered report: Diverse somatic mutation patterns and pathway alterations in human cancers

doi: 10.7554/eLife.11566

Figure Lengend Snippet:

Article Snippet: MAP2K4 WT Myc-DDK tagged –includes FLAG tag 1 , Plasmid , Origene , RC206051 , Original product number not specified.

Techniques: Cell Culture, Plasmid Preparation, Produced, Transfection, Bradford Assay, Detection Assay, Western Blot, Marker, Membrane

Journal: eLife

Article Title: Registered report: Diverse somatic mutation patterns and pathway alterations in human cancers

doi: 10.7554/eLife.11566

Figure Lengend Snippet:

Article Snippet: MAP2K4 WT Myc-DDK tagged –includes FLAG tag 1 , Plasmid , Origene , RC206051 , Original product number not specified.

Techniques: Transduction, Plasmid Preparation, Produced, Cell Culture, Software

Journal: eLife

Article Title: Registered report: Diverse somatic mutation patterns and pathway alterations in human cancers

doi: 10.7554/eLife.11566

Figure Lengend Snippet:

Article Snippet: MAP2K4 WT Myc-DDK tagged –includes FLAG tag 1 , Plasmid , Origene , RC206051 , Original product number not specified.

Techniques: Transduction, Plasmid Preparation, Produced, Cell Culture, Chromatography, Lysis, Protease Inhibitor, Magnetic Beads, Kinase Assay, Western Blot, Bradford Assay, Detection Assay, Marker, Membrane

Journal: eLife

Article Title: Registered report: Diverse somatic mutation patterns and pathway alterations in human cancers

doi: 10.7554/eLife.11566

Figure Lengend Snippet:

Article Snippet: MAP2K4 WT Myc-DDK tagged –includes FLAG tag 1 , Plasmid , Origene , RC206051 , Original product number not specified.

Techniques: Transduction

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: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Fluorofenidone protects against acute liver failure in mice by regulating MKK4/JNK pathway.

doi: 10.1016/j.biopha.2023.114844

Figure Lengend Snippet: Fig. 5. AKF-PD inhibited the early activation of MKK4/JNK. (A) Representative Western blot images of phospho-MKK4 and phospho-JNK in liver tissue. (B) Semi- quantitative analysis of phospho-MKK4 (n = 4/group). (C) Semi-quantitative analysis of phospho-JNK (n = 4/group).

Article Snippet: The antibody against GAPDH (#60004–1), MKK4 (17340–1-AP), phospho-ASK1 (28201–1-AP), ASK1 (28846–1-AP) and SH3 homology-associated BTK-binding protein (SAB, 11127–2-AP) were bought from Proteintech (Wuhan, China).

Techniques: Activation Assay, Western Blot

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Fluorofenidone protects against acute liver failure in mice by regulating MKK4/JNK pathway.

doi: 10.1016/j.biopha.2023.114844

Figure Lengend Snippet: Fig. 6. AKF-PD protects against APAP-induced acute liver injury by regulating the MKK4/JNK pathway. Primary hepatocyte isolated from mice were pretreated with culture medium or AKF-PD (400 μg/mL) for 24 h. Following this, cells were exposed to APAP (10 mM) alone or in combination with anisomycin (10 μM) or SP600125 (10 μM) for 3 h. (A) Representative Western blot images of phospho-MKK4 and phospho-JNK in primary hepatocytes. (B) Semi-quantitative analysis of phospho-MKK4 (n = 3/group). (C) Semi-quantitative analysis of phospho-JNK (n = 3/group). Ten-week-old male C57BL/6 J mice were intraperitoneally injected with anisomycin (20 mg/kg), SP600125 (15 mg/kg) or vehicle (5%DMSO), then orally administered AKF-PD (250 mg/kg) or vehicle (0.5% CMC-Na) 30 min prior to intraperitoneal injection of acetaminophen (APAP; 400 mg/kg) (n = 5/group). The liver tissue and plasma were collected 12 h after APAP challenge. (D) Repre sentative images of H&E staining in liver tissue (20 × magnification, scale bar = 500 µm and 100 × magnification, scale bar = 100 µm). (E) The liver necrosis scores of HE staining. An overall score for each slice was computed based on 5 randomly selected scopes under 100 × magnification. (F-G) ALT and AST levels in serum. (H) Representative Western blot images of phospho-MKK4 and phospho-JNK in primary hepatocytes. (I) Semi-quantitative analysis of phospho-MKK4 (n = 4/group). (K) Semi-quantitative analysis of phospho-JNK (n = 4/group). Data are represented as mean ± SEM. *p < 0.05, * *p < 0.01, and * **p < 0.001.

Article Snippet: The antibody against GAPDH (#60004–1), MKK4 (17340–1-AP), phospho-ASK1 (28201–1-AP), ASK1 (28846–1-AP) and SH3 homology-associated BTK-binding protein (SAB, 11127–2-AP) were bought from Proteintech (Wuhan, China).

Techniques: Isolation, Western Blot, Injection, Clinical Proteomics, Staining

Journal: Theranostics

Article Title: Activation of JNK and p38 MAPK Mediated by ZDHHC17 Drives Glioblastoma Multiforme Development and Malignant Progression

doi: 10.7150/thno.40076

Figure Lengend Snippet: ZDHHC17-MAP2K4-JNK/p38 Signaling Module Formation in Glioblastoma Multiforme (GBM). (A) The expression of ERK1 (pT202, pY204), ERK2 (pT185, pY187), JNK1/2(pT183, pT185), JNK3 (pT221, pY223) and p38 (pT180, pY182) in GBM is summarized based on the immunohistochemistry results of the Human Protein Atlas. (B) The expression of different DHHCs in glioma is summarized based on the immunohistochemistry results of the Human Protein Atlas. (C) Venn diagram showing the relationship between expression patterns of different DHHCs and activation of ERK1, ERK2, JNK1/2, JNK3, and p38 in GBM. (D) Lysates from HEK293 cells expressing Myc-ZDHHC17 and Flag-MAPKKs were subjected to immunoprecipitation (IP), followed up by immunoblotting (IB) with anti-FLAG antibodies and anti-Myc. IP, immunoprecipitation; IB, immunoblotting. (E) GST pull-down utilizing purified GST-MAP2K4 (or GST-MAP2K7) and FLAG-ZDHHC17-expressing HEK293 cell lysates, followed by IB with an anti-FLAG antibody. (F) ZDHHC17 associates with MAP2K4 via ZDHHC17 ANK rather than PAT activity domain. IP of lysates from HEK293 cells expressing Flag-MAP2K4 and Myc-ZDHHC17 mutants, followed by IB with anti-Flag antibodies and anti-Myc. ZDHHC17 ΔDHHC: ZDHHC17 aa 440-487 deletion; ΔANK: ZDHHC17 aa 51-288 deletion. (G) ZDHHC17 protein recruits MAP2K4 in the Golgi and cytoplasmic vesicles in U118MG cells, in a ZDHHC17 ANK domain-dependent manner. Immunofluorescence of U118MG cells expressing Myc-ZDHHC17 (or -ZDHHC17 ΔDHHC or ΔANK) with anti-Myc (Pink), MAP2K4 (Red), and GM130 (Green) antibodies. Scale bar, 20 µm. (H) ZDHHC17 interacts with MAP2K4 wild-type (wt) and the kinase-inactive mutant (ki). IP of lysates from HEK293 cells expressing Myc-ZDHHC17 and FLAG-MAP2K4wt (or FLAG-MAP2K4ki), followed by IB with anti-FLAG antibodies and anti-Myc. (I) MAP2K4 contributes to the ZDHHC7-mediated JNK1 and p38 phosphorylation. FLAG-MAP2K4ki co-expression in HEK293 cells reduces Myc-ZDHHC17-mediated GFP-JNK1 (or GFP-p38) phosphorylation. (J) MAP2K4 and JNK1 (or p38) are recruited by ZDHHC17 in a signaling module. GFP-JNK1 (p38) and FLAG-MAP2K4 were introduced in HEK293 cells, with or without Myc-ZDHHC17. MAP2K4 presence upon JNK1 (or p38) IP is improved by ZDHHC17 co-expression.

Article Snippet: The cells were transfected with Negative, MAP2K4 (#SR304323, Origene) or ZDHHC17 (#SR323571, Origene) Stealth siRNA per manufacturer instruction using Lipofectamine® RNAiMAX reagent for knockdown experimentation.

Techniques: Expressing, Immunohistochemistry, Activation Assay, Immunoprecipitation, Western Blot, Purification, Activity Assay, Immunofluorescence, Mutagenesis, Phospho-proteomics

Journal: Theranostics

Article Title: Activation of JNK and p38 MAPK Mediated by ZDHHC17 Drives Glioblastoma Multiforme Development and Malignant Progression

doi: 10.7150/thno.40076

Figure Lengend Snippet: ZDHHC17 and MAP2K4 Interact Via Specific Binding Motifs. (A) ZDHHC17 ANK (2-4) domain is crucial for ZDHHC17-MAP2K4 interaction. Myc-tagged ZDHHC17 ANK (1-7) and various deletions (right). Interaction capability (positive or negative) is shown. (B) MAP2K4 PKc domain is crucial for the ZDHHC17-MAP2K4 interaction, independent of the DVD domain. FLAG-tagged MAP2K4 and various MAP2K4 deletion fragments (right). Interaction capability (positive or negative) is shown. (C) Surface representation of the complex. ZDHHC17 and MAP2K4 binding mode molecular docking was performed using the ZDOCK server. MAP2K4 (Magenta) binds to the concave ANK(1-7) (Green) region between ANK2 and ANK4. (D) Cartoon and stick representation of ZDHHC17 ANK (1-7) (Pale Green) and ANK (1-7) (Green), respectively. (E) IP of lysates from HEK293 cells expressing Flag-MAP2K4 and Myc-ZDHHC17 ANK (1-7) mutants, followed by IB with anti-Flag antibodies and anti-Myc. Data represent the means ± SD from three separate experiments ( ns , not significant; * p < 0.05; ** p <0.01; *** p < 0.001, unpaired t- test). (F) IP of lysates from HEK293 cells expressing FLAG-MAP2K4 mutants and Myc-ZDHHC17, followed by IB with anti-Flag antibodies and anti-Myc. Data represent the means ± SD from three separate experiments ( ns , not significant; * p < 0.05; ** p < 0.01; *** p < 0.001, unpaired t -test).

Article Snippet: The cells were transfected with Negative, MAP2K4 (#SR304323, Origene) or ZDHHC17 (#SR323571, Origene) Stealth siRNA per manufacturer instruction using Lipofectamine® RNAiMAX reagent for knockdown experimentation.

Techniques: Binding Assay, Expressing

Journal: Theranostics

Article Title: Activation of JNK and p38 MAPK Mediated by ZDHHC17 Drives Glioblastoma Multiforme Development and Malignant Progression

doi: 10.7150/thno.40076

Figure Lengend Snippet: ZDHHC17 and MAP2K4 Expression Correlates with Glioma Tumor Grade and has Prognostic Significance . (A) Representative images of immunohistochemistry staining for ZDHHC17 and MAP2K4 in different grade gliomas and normal brain specimens. Scale bar, 100 µm. (B, C) Correlation between MAP2K4 and ZDHHC17 (B) or ZDHHC13 (C) in GBM using TCGA datasets. Correlation statistical significance was evaluated using a linear regression model (n = 171, r = 0.4394, p < 0.001) (B), (n = 172, r = -0.07494, p = 0.3286) (C). (D-G) Accumulative overall (D; F, n=164) and disease-free survival (E; G, n=118) of patients with high or low ZDHHC17 (D, E) or MAP2K4 (F, G) expression GBM (based on IHC staining results) calculated using the Kaplan-Meier method and compared with the log-rank test for the same set of patients (* p < 0.05; ** p < 0.01).

Article Snippet: The cells were transfected with Negative, MAP2K4 (#SR304323, Origene) or ZDHHC17 (#SR323571, Origene) Stealth siRNA per manufacturer instruction using Lipofectamine® RNAiMAX reagent for knockdown experimentation.

Techniques: Expressing, Immunohistochemistry, Staining

Journal: Theranostics

Article Title: Activation of JNK and p38 MAPK Mediated by ZDHHC17 Drives Glioblastoma Multiforme Development and Malignant Progression

doi: 10.7150/thno.40076

Figure Lengend Snippet: Genistein is a Lead Candidate for Inhibiting the ZDHHC17-MAP2K4 Interaction in GBM. (A) GBM0378 and GBM1492, from patients with GBM, cell viability after 24h-treatment with indicated inhibitors at indicated concentration from the MAPK Compound Library. Data represent the means ± SD from three separate experiments (** p < 0.01; *** p < 0.001, unpaired t -test). (B, C) Western blot and quantitation of JNK1/2 and p38 phosphorylation; β-actin was used as a loading control (B), and immunoprecipitation (IP) of the ZDHHC17-MAP2K4 interaction (C) after 24h-treatment with indicated inhibitors (5.0 μM for each inhibitor) in ZDHHC17-expressing U118MG cells. Data represent the means ± SD from three separate experiments (* p < 0.05; ** p < 0.01; *** p < 0.001, unpaired t -test). (D) Genistein treatment directly inhibits ZDHHC17-MAP2K4 kinase activity. ZDHHC17-MAP2K4 kinase activity in the presence of genistein (0-80 μM) measured with commercially available kits using a passive JNK2 as the substrate. The phosphorylated substrate was tested by western blot and band density calculated using AlphaEaseFc software. Data represent the percentage of activity in the untreated control reaction; the means ± SD from three separate experiments are shown (* p < 0.05; ** p < 0.01, unpaired t -test). (E) Surface representation of the complex. Molecular docking of the binding mode between ZDHHC17 (Green) and genistein was performed using the ZDOCK server.

Article Snippet: The cells were transfected with Negative, MAP2K4 (#SR304323, Origene) or ZDHHC17 (#SR323571, Origene) Stealth siRNA per manufacturer instruction using Lipofectamine® RNAiMAX reagent for knockdown experimentation.

Techniques: Concentration Assay, Drug discovery, Western Blot, Quantitation Assay, Phospho-proteomics, Control, Immunoprecipitation, Expressing, Activity Assay, Software, Binding Assay

Journal: Theranostics

Article Title: Activation of JNK and p38 MAPK Mediated by ZDHHC17 Drives Glioblastoma Multiforme Development and Malignant Progression

doi: 10.7150/thno.40076

Figure Lengend Snippet: ZDHHC17-MAP2K4 Signaling Module is Necessary for GBM Cell Tumorigenic and Invasive Phenotypes. (A, B) Clonogenic survival of (A) U118MG cells transfected with control or ZDHHC17 dsRNA, further transfected with ZDHHC17 plasmid or MAP2K4 dsRNA, or treated with genistein (2.5 μM), and (B) SW1088 cells transfected with control or ZDHHC17 plasmid, further transfected with ZDHHC17 dsRNA or MAP2K4 plasmid, or genistein (2.5 μM) treated and ZDHHC17-expressing. Data represent the means ± SD from three separate experiments ( ns , non-significant; * p < 0.05; ** p < 0.01; *** p < 0.001, unpaired t -test). (C-F) Transwell analysis of (C, E) migratory and (D, F) invasive U118MG cells transfected with control or ZDHHC17 dsRNA, or further transfected with ZDHHC17 plasmid or MAP2K4 dsRNA (C, D), and SW1088 cells transfected with control or ZDHHC17 plasmid, or further transfected with ZDHHC17 dsRNA or MAP2K4 plasmid (E, F). Data represent the means ± SD from three separate experiments ( ns , not significant; ** p < 0.01; *** p < 0.001, unpaired t -test). Scale bar, 500 μm. (G, H) Cell cycle of U118MG cells transfected with control or ZDHHC17 dsRNA, further transfected with ZDHHC17 plasmid or MAP2K4 dsRNA, or genistein (2.5 μM)-treated, as detected by flow cytometry. The percent of cells in the G0-G1 phase, S-phase, and G2-M phase was calculated. Data represent the means ± SD from three separate experiments (* p < 0.05; ** p < 0.01; *** p < 0.001, unpaired t -test).

Article Snippet: The cells were transfected with Negative, MAP2K4 (#SR304323, Origene) or ZDHHC17 (#SR323571, Origene) Stealth siRNA per manufacturer instruction using Lipofectamine® RNAiMAX reagent for knockdown experimentation.

Techniques: Transfection, Control, Plasmid Preparation, Expressing, Flow Cytometry

Journal: Theranostics

Article Title: Activation of JNK and p38 MAPK Mediated by ZDHHC17 Drives Glioblastoma Multiforme Development and Malignant Progression

doi: 10.7150/thno.40076

Figure Lengend Snippet: ZDHHC17-MAP2K4 Signaling Module is Required for GBM Cell Neurosphere Formation. (A-C) Flow cytometry and quantitation of SOX2 (stem cell marker) (A, B) and western blot of SOX2, CD133, and Nestin using β-actin as a loading control (C) in SW1088 cells transfected with ZDHHC17 plasmid or control, further transfected with MAP2K4 dsRNA, or genistein (2.5 μM)-treated. Data represent the means ± SD from three separate experiments (*** p < 0.001, unpaired t -test). (D) Self-renewal of glioma stem cells (GSCs) originating from U118MG cells transfected with ZDHHC17 or MAP2K4 dsRNA, or genistein (2.5 μM) or 2-bromopalmitate (palmitoylation inhibitor; 200 μM)-treated, by neurosphere formation assay. Neurosphere numbers from dsRNA-transduced or inhibitor-treated ZDHHC17-expressing GSCs were calculated. Data represent the means ± SD from three separate experiments ( ns , not significant; *** p < 0.001, unpaired t -test). (E) Neurosphere capacity by serial dilution assay of GSCs from U118MG cells transfected with ZDHHC17 or MAP2K4 dsRNA, or genistein (2.5 μM) or 2-bromopalmitate (200 μM)-treated. Data represent the means ± SD from three separate experiments ( ns, not significant; *** p < 0.001, unpaired t -test). (F) Colonies formed by 200 viable GSCs from U118MG cells transfected with MAP2K4 dsRNA or ZDHHC17, or genistein (2.5 μM) or 2-bromopalmitate (200 μM)-treated. Data represent the means ± SD from three separate experiments ( ns , not significant; *** p < 0.001, unpaired t -test). (G) Representative photon flux images from BALB/c mouse brains implanted with luciferase-expressing GSCs from U118MG cells transfected with control (left) or ZDHHC17 shRNA (right), then further genistein (50 mg kg -1 two days -1 ) or 2-bromopalmitate (100 mg kg -1 day -1 )-treated. (H) Bioluminescence signal intensity in intracranial tumors between control (left) or ZDHHC17 shRNA (right) U118MG GSCs with or without 2-bromopalmitate or genistein (n = 5). Data represent the means ± SD ( ns , not significant; * p < 0.05; ** p < 0.01; *** p < 0.001, unpaired t -test). (I) Kaplan-Meier survival curve of animal survival following control or ZDHHC17 shRNA U118MG GSC cell injection with or without genistein or 2-bromopalmitate.

Article Snippet: The cells were transfected with Negative, MAP2K4 (#SR304323, Origene) or ZDHHC17 (#SR323571, Origene) Stealth siRNA per manufacturer instruction using Lipofectamine® RNAiMAX reagent for knockdown experimentation.

Techniques: Flow Cytometry, Quantitation Assay, Marker, Western Blot, Control, Transfection, Plasmid Preparation, Tube Formation Assay, Expressing, Serial Dilution Assay, Luciferase, shRNA, Injection

Journal: Theranostics

Article Title: Activation of JNK and p38 MAPK Mediated by ZDHHC17 Drives Glioblastoma Multiforme Development and Malignant Progression

doi: 10.7150/thno.40076

Figure Lengend Snippet: ZDHHC17-MAP2K4 Signaling Module Promotes Chemoradiotherapy Resistance in GBM Spheres. (A) mRNA expression analysis (Mao's dataset, GSE67089) of ZDHHC17 expression in mesenchymal (MES) GSCs compared to normal astrocytes, or proneural (PN) GSCs. (B) Genome-wide transcriptome microarray analysis (GSE67089) of MAPKKs showing MAP2K4 up-regulation in MES compared with PN GSCs. (C) RT-PCR for ZDHHC17 and MAP2K4 in post-radiation GSCs from U118MG (6 Gy) or temozolomide (TMZ)-treated (25 μM) versus naive GSCs. (D) Western blot for ZDHHC17, MAP2K4, and EZH2 in post-radiation GSCs from U118MG (6 Gy) or TMZ-treated GSCs (25 μM) versus naive GSCs. (E) Flow cytometric analysis for apoptosis in GSCs pre-transduced with control or ZDHHC17 dsRNA, then treated with or without genistein (2.5 μM), radiation (6 Gy), and TMZ (25 μM). (F) BALB/c mice were subcutaneously injected with GSCs from U118MG. After five days, the nude mice were treated with 20 Gy X-irradiation (4.5-4.6 Gy/min), TMZ (50 mg kg -1 two days -1 , gastric infusion), or genistein (100 mg/kg daily, tail vein injection). Tumor weight was quantified. Data represent the means ± SD from five separate experiments ( ns , not significant; ** p < 0.01; *** p < 0.001, unpaired t -test). (G) Typical immunohistochemistry images for ZDHHC17 and MAP2K4 in primary untreated and post-radiation and -TMZ chemotherapy recurrent tumors from matched patients with GBM. Scale bars, 200 µm. (H) Percentage of ZDHHC17 or MAP2K4 positive-stained cells in primary untreated and recurrent tumors from matched patients with GBM (n = 5; * p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: The cells were transfected with Negative, MAP2K4 (#SR304323, Origene) or ZDHHC17 (#SR323571, Origene) Stealth siRNA per manufacturer instruction using Lipofectamine® RNAiMAX reagent for knockdown experimentation.

Techniques: Expressing, Genome Wide, Microarray, Reverse Transcription Polymerase Chain Reaction, Western Blot, Transduction, Control, Injection, Irradiation, Immunohistochemistry, Staining

Journal: Molecular and Cellular Biology

Article Title: Map2k4 Functions as a Tumor Suppressor in Lung Adenocarcinoma and Inhibits Tumor Cell Invasion by Decreasing Peroxisome Proliferator-Activated Receptor γ2 Expression

doi: 10.1128/mcb.05562-11

Figure Lengend Snippet: FIG. 1. MAP2K4 mutations in human cancers and sequence alignment of MKK family members. (A) MAP2K4 mutations. Eleven MAP2K4 mutations found in human cancers analyzed herein are depicted schematically and in tabular form, denoting the cancer types associated with them (which are referenced) and the primers used to create these mutants by site-directed mutagenesis (mutation sites are underlined and in bold). The synthetic MAP2K4 dominant-active S257E; T261D (ED) mutant and dominant-negative K131R (KR) mutant used as controls in this study are indicated. *, nonsense mutation; fs, frameshift mutation. (B) Amino acid sequences in MKK family members were aligned using the ClustalW method. MAP2K4 mutation sites are labeled and boxed. Certain mutation sites (N234, S251, and P326) are well conserved across MKK family members.

Article Snippet: Vectors expressing mouse Map2k4 (OriGene), Bgn, Vegfc, Satb1, Pparg (SA Biosciences), MAP2K4, and PPARG short hairpin RNAs (shRNAs) were purchased (Open Biosystems).

Techniques: Sequencing, Mutagenesis, Dominant Negative Mutation, Labeling

Journal: Molecular and Cellular Biology

Article Title: Map2k4 Functions as a Tumor Suppressor in Lung Adenocarcinoma and Inhibits Tumor Cell Invasion by Decreasing Peroxisome Proliferator-Activated Receptor γ2 Expression

doi: 10.1128/mcb.05562-11

Figure Lengend Snippet: FIG. 2. MAP2K4 mutants exhibit aberrant kinase activities. (A) MKK4 mutation sites were superimposed on an MKK4 X-ray crystal structure (Protein Data Bank; 3ALO) using PyMOL (http://www.pymol.org). Mutated amino acids and their locations are indicated with arrows and labeled in red. (B) Prediction algorithms were used to predict whether MAP2K4 missense mutations affect kinase activity (PolyPhen and SIFT) and are associated with cancer (CanPredict). (C) 293T cells were transfected with Flag-tagged MKK4 mutants and then immunoprecipitated (IP) with Flag antibody. In vitro kinase assays were performed using a kinase-dead mutant form (K55M) of JNK1 as a substrate. As controls, the synthetic MAP2K4 dominant-active S257E; T261D (ED) and dominant-negative K131R (KR) mutants were used. KA, kinase activity; WB, Western blot; total WB, WB of total lysate. (D) Quantification of kinase assays. The activity of each mutant was normalized to that of the wild type (WT), which was set at a value of 1. Graph values are presented as means standard deviations (SD) (n 3), and P values were calculated with Student’s t test (* or #, P 0.01). (E) Response of mutants to stress. 293T cells were transfected with Flag-MKK4 alone and treated with UV (250 J/m2 for 30 min) or with sorbitol (0.5 M for 3 h). Kinase assays performed as described above. (F) Response of mutants to upstream signal. 293T cells were cotransfected with Flag-MKK4 mutants and MEKK1 (an upstream kinase of MKK4), and then in vitro kinase assays were performed.

Article Snippet: Vectors expressing mouse Map2k4 (OriGene), Bgn, Vegfc, Satb1, Pparg (SA Biosciences), MAP2K4, and PPARG short hairpin RNAs (shRNAs) were purchased (Open Biosystems).

Techniques: Mutagenesis, Labeling, Activity Assay, Transfection, Immunoprecipitation, In Vitro, Dominant Negative Mutation, Western Blot

Journal: Molecular and Cellular Biology

Article Title: Map2k4 Functions as a Tumor Suppressor in Lung Adenocarcinoma and Inhibits Tumor Cell Invasion by Decreasing Peroxisome Proliferator-Activated Receptor γ2 Expression

doi: 10.1128/mcb.05562-11

Figure Lengend Snippet: FIG. 3. C-terminally truncated MAP2K4 mutants are rapidly degraded. (A) 293T cells were transfected with MAP2K4 mutants and subjected to Western blotting using anti-MKK4 or antitubulin. The protein levels of truncated forms (C-term) were very low compared with those of full-length forms. (B) Reverse transcriptase PCR of Flag-tagged MKK4 mRNA. There was no difference in mRNA expression levels between wild-type and C-terminally truncated MKK4. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (C) 35S-Met labeling to measure protein stability, demonstrating the extinction of the truncated mutant forms by 4 h after the pulse. (D) Quantification of data in panel C by densitometric analysis. (E) In vivo ubiquitination assay. Immunoprecipitation and Western blotting of transfectants revealed that truncated forms were more highly ubiquitinated than were full-length forms. A total of 2-fold more lysates of the truncated mutants were loaded to compensate for their rapid degradation. (F) Proteasome inhibitors MG132 (50 M) and -lactone (clasto-lactacystin--lactone) (5 M) attenuated the degradation of C-terminally truncated mutants based on 35S-Met labeling analyses. (G) Deletion of 300 (300del) but not 100 (100del) or 200 (200del) C-terminal amino acids abrogated ubiquitination of MKK4. MKK4 C-terminal deletion constructs were transiently transfected into 293T cells, immunoprecipitated using anti-Flag antibodies, and subjected to Western blotting. The numbers of N-terminal amino acids remaining in the deletion constructs are indicated on right side of the panel.

Article Snippet: Vectors expressing mouse Map2k4 (OriGene), Bgn, Vegfc, Satb1, Pparg (SA Biosciences), MAP2K4, and PPARG short hairpin RNAs (shRNAs) were purchased (Open Biosystems).

Techniques: Transfection, Western Blot, Reverse Transcription, Expressing, Labeling, Mutagenesis, In Vivo, Ubiquitin Proteomics, Immunoprecipitation, Construct

Journal: Molecular and Cellular Biology

Article Title: Map2k4 Functions as a Tumor Suppressor in Lung Adenocarcinoma and Inhibits Tumor Cell Invasion by Decreasing Peroxisome Proliferator-Activated Receptor γ2 Expression

doi: 10.1128/mcb.05562-11

Figure Lengend Snippet: FIG. 4. MKK4 inactivation enhances lung tumorigenesis driven by mutant Kras. (A) Map2k4 inactivation shortened mouse survival times. The graph presents the results of Kaplan-Meier survival analysis of mice with the indicated genotypes. P values for comparison of the results obtained with KrasG12D/ and KrasG12D/ ; MKK4L/L groups were calculated with the log rank (Mantel-Cox) test. (B) Recombination of KrasLSL allele specifically in lung tumors. KrasWT, wild-type Kras allele ( 622 bp); KrasLSL, germ line conditional Kras allele (500 bp); KrasG12D, recombined conditional Kras allele ( 650 bp). The PCR amplification strategy employed has been previously reported (20). (C) PCR confirmation of floxed allele recombination. A diagrammatic illustration of the PCR amplification strategy (top) used to detect the wild-type Map2k4 allele (MKK4 ) and germ line allele (MKK4L) and recombined (MKK4 ) floxed Map2k4 allele by the use of forward (F) and reverse (R) primers is shown. The left gel demonstrates recombination of MKK4L specifically in lung tissues of KrasG12D/ ; MKK4L/L mice. The right gel demonstrates recombination of the MKK4L allele specifically in lung tumors. The PCR amplification strategy used has been previously reported (46). (D) Histologic sections of lung tissues. Lung tissue sections from each cohort were stained with hematoxylin and eosin and photographed (main images). Magnifications of adenomas are shown in the insets. (E) Examples of lung tissues obtained at necropsy. Lungs and heart (H) were removed at necropsy. Photographs illustrate representative lung tissues that are wild type (WT) or have inactivated Map2k4 (MKK4L/L) or express KrasG12D or both. Lung tumors (arrows) are indicated. (F and G) Map2k4 inactivation promoted lung tumor growth. The numbers of lung tumors visible on the pleural surface were determined at necropsy and are expressed as the mean value per lung per cohort (F). Areas of tumor surface in lung tissue sections were measured using ImageJ (http://rsbweb.nih.gov/ij) and are expressed as the ratio of the sum of all tumor surface areas to lung surface area per cohort (G). Mean values, long horizontal lines; standard deviations, short horizontal lines.

Article Snippet: Vectors expressing mouse Map2k4 (OriGene), Bgn, Vegfc, Satb1, Pparg (SA Biosciences), MAP2K4, and PPARG short hairpin RNAs (shRNAs) were purchased (Open Biosystems).

Techniques: Mutagenesis, Comparison, Staining

Journal: Molecular and Cellular Biology

Article Title: Map2k4 Functions as a Tumor Suppressor in Lung Adenocarcinoma and Inhibits Tumor Cell Invasion by Decreasing Peroxisome Proliferator-Activated Receptor γ2 Expression

doi: 10.1128/mcb.05562-11

Figure Lengend Snippet: FIG. 5. MKK4 depletion enhances tumor cell migration and invasion. (A) Flag-tagged MKK4 (wild-type or mutant) vectors (vec) were stably transfected into 344SQ cells. (B) MKK4-transfected 344SQ cells were counted every day for 5 days under normal culture conditions. Wild-type MKK4 had no effect, but a constitutively active (ED) mutant slightly inhibited cellular growth. Results represent the mean values (SD) obtained with quadruplicate wells. *, P 0.05; **, P 0.01 (compared with vector). (C) RAW 264.7- and MKK4-transfected 344SQ cells were seeded into the lower and upper chambers, respectively, of Matrigel-coated chambers for invasion assays. Invasive cells were photographed (images) and counted (bar graph). Results represent the mean values (SD) obtained with triplicate wells. (D) 393P cells were transfected with each of four distinct MKK4 shRNAs (A, B, Y, or Z) and subjected to Western blotting, which was quantified densitometrically relative to the results obtained with a scrambled control, which was set at a value of 1. Two clones with the most prominent MKK4 depletion (shY and shZ) were selected for further experiments. (E and F) 393P-scr, shY, and shZ cells were seeded into 12-well plates, and cell numbers were counted every day for 5 days under normal culture conditions (E) or after 2 days under serum-free conditions (0%) (F), revealing a slight increase in cellular proliferation under normal conditions at days 2 and 3 but not at later time points. *, P 0.05 compared with scr results. (G and H) MKK4 depletion enhanced 393P cell migration and invasion. RAW 264.7 and 393P cells were seeded into the lower and upper chambers, respectively, of Transwell plates for migration assays (G) and Matrigel-coated chambers for invasion assays (H) in the presence of mitomycin C (1 g/ml). Cells were photographed (images) and counted (bar graphs). Results represent mean values (SD) obtained with triplicate wells. (I) H2009 human lung cancer cells were stably transfected with each of five distinct human MKK4 shRNAs (sh1 to sh5). The two clones with the most prominent MKK4 depletion (sh3 and sh4) were selected for further experiments. (J) H2009 transfectants (scr, sh3, and sh4) were counted every day for 5 days under normal culture conditions, which revealed increased proliferation in clone sh3. Results represent mean values (SD) obtained with triplicate wells. *, P 0.05; **, P 0.01 (compared with scr results). (K) H2009 transfectants were seeded into Matrigel-coated chambers for invasion assays. Invasive cells were photographed (images) and counted (bar graph). Results represent mean values (SD) obtained with triplicate wells.

Article Snippet: Vectors expressing mouse Map2k4 (OriGene), Bgn, Vegfc, Satb1, Pparg (SA Biosciences), MAP2K4, and PPARG short hairpin RNAs (shRNAs) were purchased (Open Biosystems).

Techniques: Migration, Mutagenesis, Stable Transfection, Transfection, Plasmid Preparation, Western Blot, Control, Clone Assay

Journal: Molecular and Cellular Biology

Article Title: Map2k4 Functions as a Tumor Suppressor in Lung Adenocarcinoma and Inhibits Tumor Cell Invasion by Decreasing Peroxisome Proliferator-Activated Receptor γ2 Expression

doi: 10.1128/mcb.05562-11

Figure Lengend Snippet: FIG. 6. MKK4 reintroduction abrogates invasive activity. (A) Flag-tagged MKK4 (wild-type or mutant) vectors were stably transfected into 393P-shZ cells. (B and C) 393P-shZ-MKK4 cells and RAW 264.7 cells were seeded into the upper and lower chambers, respectively, of Matrigel-coated wells, and then invasive cells were photographed (B) and counted (C). Results represent mean values (SD) obtained with triplicate wells. *, P 0.01 compared to empty vector. (D) Graph denoting the relationship between MKK4 kinase activity and invasion activity. According to linear regression analysis, most MKK4 mutants were within 95% confidence intervals (dotted curves; R2 0.6737). (E) Flag-tagged MKK4 (wild-type or S251N mutant) vectors were stably transfected into BxPC3 cells. (F) Cell proliferation of MKK4-transfected BxPC3 cells was measured by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assays daily for 4 days. Wild-type MKK4 had no effect, whereas the S251N mutant increased cellular growth. Results represent mean values (SD) obtained with quadruplicate wells. *, P 0.01 compared with vector. (G) Wild-type MKK4 (WT) suppressed BxPC3 cell invasion in Matrigel-coated chambers, whereas the S251N mutant slightly increased invasion. Invasive cells were photographed and counted (bar graph). Results are expressed as mean values (SD) obtained with triplicate wells. (H) Inhibitors of JNK (SP) or p38 (SB) blocked the phosphorylation of downstream targets but did not increase 393P cell invasive

Article Snippet: Vectors expressing mouse Map2k4 (OriGene), Bgn, Vegfc, Satb1, Pparg (SA Biosciences), MAP2K4, and PPARG short hairpin RNAs (shRNAs) were purchased (Open Biosystems).

Techniques: Activity Assay, Mutagenesis, Stable Transfection, Transfection, Plasmid Preparation, Phospho-proteomics

Journal: Molecular and Cellular Biology

Article Title: Map2k4 Functions as a Tumor Suppressor in Lung Adenocarcinoma and Inhibits Tumor Cell Invasion by Decreasing Peroxisome Proliferator-Activated Receptor γ2 Expression

doi: 10.1128/mcb.05562-11

Figure Lengend Snippet: FIG. 7. Expression profiling of 393P-shZ and 393P-scr cells. (A) Expression data matrix (“heat map”) of 574 RNA transcripts (i.e., Affymetrix probe sets, representing 449 unique genes) differentially expressed between 393P-shZ (MKK4-shRNA) and 393P-scr (control). Each row represents a gene, each column a profiled sample. The relative abundance of each gene in each group is represented using a yellow-blue color scale (blue represents low expression; yellow represents high expression). To the right of the data matrix, red, green, blue, and yellow bars denote the corresponding annotations of genes in the matrix presented using selected Gene Ontology (GO) terms. Specific genes of particular interest

Article Snippet: Vectors expressing mouse Map2k4 (OriGene), Bgn, Vegfc, Satb1, Pparg (SA Biosciences), MAP2K4, and PPARG short hairpin RNAs (shRNAs) were purchased (Open Biosystems).

Techniques: Expressing, shRNA, Control

Journal: Molecular and Cellular Biology

Article Title: Map2k4 Functions as a Tumor Suppressor in Lung Adenocarcinoma and Inhibits Tumor Cell Invasion by Decreasing Peroxisome Proliferator-Activated Receptor γ2 Expression

doi: 10.1128/mcb.05562-11

Figure Lengend Snippet: FIG. 8. MKK4 suppresses tumor cell invasion through PPAR. (A) Quantitative RT-PCR (Q-PCR) analysis of PGC-1 mRNA in 393P-scr and 393P-shZ cells. Data represent mean values (SD) obtained with triplicate samples. (B) Q-PCR analyses of PPAR2 (left) and PGC-1 (middle) and reporter assays (right) in cells transiently transfected with a PPAR-responsive element (PPRE) luciferase reporter plasmid. Results were normalized on the basis of renilla luciferase activity values and are expressed as mean values (SD) obtained with triplicate samples. (C and D) Q-PCR analyses of PPAR2 mRNA in 344SQ cells (C) or 393P-shZ cells (D) stably transfected with shRNAs against PPAR (shP-A or shP-B) or scrambled control (scr). Results were normalized on the basis of L32 mRNA values and are expressed as mean values (SD) obtained with triplicate samples. (E and F) Invasion assays on 344SQ cells (E) or 393P-shZ cells (F) stably transfected with PPAR or scrambled shRNA. Invasive cells were photographed (images) and quantified (bar graphs). Results are expressed as mean values (SD) obtained with triplicate wells. (G) Invasion assays on 393P-shZ cells treated with a PPAR-selective antagonist (T0070907) or vehicle (DMSO). Invaded cells were photographed (images) and quantified (bar graph). Vehicle-treated cells are indicated as a “0” dose. Cellular toxicity after 24 h of treatment of 393P-shZ cells with T0070907 was examined by MTT assay (line graph). Vehicle-treated cells are indicated as a “0” dose. Results are expressed as mean values (SD) obtained with triplicate wells. (H) Invasion assays on 393P cells stably transfected with PPAR cDNA or empty (vec) expression vectors. Invasive cells were photographed (images) and quantified (left bar graph). Results are expressed as mean values (SD) obtained with triplicate wells. Q-PCR analysis confirmed exogenous PPAR mRNA expression (right bar graph). Results were normalized on the basis of L32 mRNA values and are expressed as mean values (SD) obtained with triplicate samples. (I) Q-PCR analysis of human pancreatic cancer cells (BxPC3) stably transfected with one of four distinct shRNAs against PPAR (shP1 to shP4) or scrambled control (scr). Results were normalized on the basis of L32 mRNA values and are expressed as mean values (SD) obtained with triplicate samples. (J) Invasion assays on BxPC3 cells stably transfected with PPAR shRNAs (shP3 and shP4) or scrambled (scr) shRNA. Invasive cells were photographed (images) and quantified (bar graphs). Results are expressed as mean values (SD) obtained with triplicate wells.

Article Snippet: Vectors expressing mouse Map2k4 (OriGene), Bgn, Vegfc, Satb1, Pparg (SA Biosciences), MAP2K4, and PPARG short hairpin RNAs (shRNAs) were purchased (Open Biosystems).

Techniques: Quantitative RT-PCR, Transfection, Luciferase, Plasmid Preparation, Activity Assay, Stable Transfection, Control, shRNA, MTT Assay, Expressing

Journal: Scientific Reports

Article Title: Prevention of calpain-dependent degradation of STK38 by MEKK2-mediated phosphorylation

doi: 10.1038/s41598-019-52435-8

Figure Lengend Snippet: MEKK2 inhibits heat- and calpain-mediated degradation of STK38. ( A ) COS-7 cells were transfected with human STK38-V5 alone, or with FLAG-MEKK1 or FLAG-MEKK2. Forty-eight hours after transfection, the cells were heated to 44 °C for 20 min or left untreated as controls and harvested. ( B ) COS-7 cells were transfected with human STK38-V5 alone, or with FLAG-MEKK2 (WT) or FLAG-MEKK2 (KM). Forty-eight hours after transfection, the cells were harvested. ( C ) HeLa cells were transfected with the scramble oligonucleotides control (scr) or MEKK2 -specific siRNA. Forty-eight hours after transfection, the cells were heated to 44 °C for the indicated times or left untreated as controls and harvested. ( D ) HeLa cells were transfected with FLAG-MEKK2. Forty-eight hours after transfection, the cells were heated to 44 °C for the indicated times or left untreated as controls and harvested. The MEKK2 activity was measured by immune complex kinase assay with an anti-FLAG antibody using GST-MKK4 as the substrate. ( E ) GST-STK38 was incubated with calpain I (0.07 units of calpain I in each lane) in the absence or presence of GST-active MEKK2 for 15 min at 30 °C. Cell lysates or in vitro reaction products were analysed by western blotting with the antibodies against the indicated proteins. A representative image of western blot is shown (see Supplementary Fig for corresponding full-length image). Relative levels of STK38 were determined from the western blot using Image J software. Data are presented as the mean ± standard deviation of three independent experiments. Statistical significance was determined by the Student’s t -test (** P < 0.05).

Article Snippet: For the MEKK2 kinase assay, V5-STK38 immunoprecipitates were incubated with 10 ng of active-MEKK2 (Signal Chem, Richmond, BC) in MEKK2 kinase buffer containing 0.37 MBq ml −1 [γ- 32 P] ATP at 30 °C for 15 min. For in vitro kinase assays, active MEKK2 was incubated with 1.0 μg of wild-type inactive STK38 (Signal Chem) or inactive MKK4 (Signal Chem) in MEKK2 kinase buffer containing 0.37 MBq ml −1 [γ- 32 P] ATP for 30 min at 30 °C.

Techniques: Transfection, Control, Activity Assay, Immune Complex Kinase Assay, Incubation, In Vitro, Western Blot, Software, Standard Deviation

Journal: Scientific Reports

Article Title: Prevention of calpain-dependent degradation of STK38 by MEKK2-mediated phosphorylation

doi: 10.1038/s41598-019-52435-8

Figure Lengend Snippet: Maintenance of STK38 stability requires its phosphorylation. ( A ) MEKK2 phosphorylates STK38 in vitro . GST-tagged STK38 (unactive) or MKK4 (unactive) was incubated with or without active MEKK2 in the presence of [γ- 32 P] ATP for 30 min at 30 °C. ( B , upper) Putative phosphorylation sites identified within STK38. Inactive GST-STK38 was incubated with or without active MEKK2, and the kinase reaction products were subjected to SDS-PAGE. GST-STK38 was excised and processed by tryptic cleavage for MS analysis. *T74 is endogenously phosphorylated. ( B , bottom) In vitro kinase reaction was performed by incubating active MEKK2 alone or with the V5-immunopurified wild-type STK38, STK38 (S91A), STK38 (T243A), STK38 (T270A) from the transfected 293 T cells for 15 min at 30 °C. The kinase reaction products were subjected to SDS-PAGE and then visualised by autoradiography ( 32 P, top panel) or Coomassie Brilliant Blue staining (CBB, bottom panel). ( C ) COS-7 cells were transfected with human STK38-V5 (WT) or STK38-V5 (S91A). Forty-eight hours after transfection, the cells were harvested. In vitro cleavage reaction was performed by incubating calpain I (0.07 units) with lysates (30 μg) of the transfected cells from STK38-V5 (WT) or STK38-V5 (S91A) for 30–60 min at 30 °C. Reaction products were subjected to western blotting analysis with antibodies against the indicated proteins. ( D ) COS-7 cells were transfected with human STK38-V5 (WT) or STK38-V5 (S91A). Forty-eight hours after transfection, the cells were treated as described in Fig. . Cell lysates were analysed by western blotting with antibodies against the indicated proteins. A representative image of western blot, CBB-stained gel, or autoradiography is shown (see Supplementary Fig for corresponding full-length image). Relative levels of STK38 were determined from the western blot by using Image J software. Data are presented as the mean ± standard deviation of three independent experiments. Statistical significance was determined by the Student’s t -test (* P < 0.05; ** P < 0.01).

Article Snippet: For the MEKK2 kinase assay, V5-STK38 immunoprecipitates were incubated with 10 ng of active-MEKK2 (Signal Chem, Richmond, BC) in MEKK2 kinase buffer containing 0.37 MBq ml −1 [γ- 32 P] ATP at 30 °C for 15 min. For in vitro kinase assays, active MEKK2 was incubated with 1.0 μg of wild-type inactive STK38 (Signal Chem) or inactive MKK4 (Signal Chem) in MEKK2 kinase buffer containing 0.37 MBq ml −1 [γ- 32 P] ATP for 30 min at 30 °C.

Techniques: Phospho-proteomics, In Vitro, Incubation, SDS Page, Transfection, Autoradiography, Staining, Western Blot, Software, Standard Deviation

Journal: Contrast Media & Molecular Imaging

Article Title: Lidocaine Ameliorates Diabetic Peripheral Neuropathy in Streptozotocin-Induced Diabetic Rats through Modulating the c-Jun Signaling Pathway

doi: 10.1155/2022/1888153

Figure Lengend Snippet: Primer sequences.

Article Snippet: After enclosing with 5% skimmed milk, the antibodies including anti-TNF- α (bs-10802R, 1 : 2, 000, Bioss, China), anti-IL-6 (bs-4539R, 1 : 2, 000, Bioss, China), anti-MKK4 (bs-1977R, 1 : 2, 000, Bioss, China), anti-p-MKK4 (bs-3392R, 1 : 2, 000, Bioss, China), anti-p-JNK (bsm-52462R, 1 : 2, 000, Bioss, China), anti-p-c-Jun (bs-12913R, 1 : 2, 000, Bioss, China), and anti-GAPDH (bs-0755R, 1 : 2, 000, Bioss, China) are incubated overnight.

Techniques: