Journal: Journal of advanced research

Article Title: Hypoxia-induced degradation of FTO promotes apoptosis by unmasking RACK1-mediated activation of MTK1-JNK1/2 pathway.

doi: 10.1016/j.jare.2025.01.019

Figure Lengend Snippet: Fig. 8. Binding between FTO and RACK1 impedes the association of RACK1 with MTK1 and JNK1/2, thereby preventing MTK1 from phosphorylating JNK1/2. A and B, NIH/3T3 cells were co-transfected with fto siRNA, RACK1-FLAG, FTO-MYC, or truncated FTO (FTO-MYC) variants (1–167 aa, 168–334 aa, or 335–502 aa) for 12 h. Cells were then collected to determine the protein levels of FLAG, MYC, JNK1/2, and MTK1 by Western blot. Immunoprecipitation assays were performed to analyze the interactions between RACK1 and FTO, RACK1 and MTK1, and MTK1 and JNK1/2. C and D, NIH/3T3 cells transfected with atg7 siRNA (C) or FTO-WT-MYC (D) for 12 h were cultured for an additional 12 h under normoxic (21 % O2) or hypoxic (1 % O2) conditions. Cells were then collected to determine the protein levels of p-JNK1/2 (Thr183/Tyr185) and JNK1/2 by Western blot. E–G, NIH/3T3 cells were co-transfected with fto siRNA, RACK1-FLAG, FTO-MYC, truncated FTO (FTO-MYC) variants, or mtk1 siRNA for 12 h. Protein levels of p-JNK1/2 (Thr183/Tyr185) and JNK1/2 were determined by Western blot. H, NIH/3T3 cells transfected with pcDNA 3.1, FTO-MYC (335–502 aa), FTO-MYC (1–167 aa), or mtk1 siRNA for 12 h were cultured for an additional 24 h under normoxia. Protein levels of FLAG, MYC, p-JNK1/2 (Thr183/Tyr185), and MTK1 were measured by Western blot. Immunoprecipitation assays were performed to analyze the binding of JNK1/2 with FLAG and MTK1.

Article Snippet: Antibodies against RACK1 (27592–1-AP), NEDD4 (21698–1-AP), and MTK1 (21610–1-AP) were obtained from Proteintech.

Techniques: Binding Assay, Transfection, Western Blot, Immunoprecipitation, Cell Culture

Journal: Journal of advanced research

Article Title: Hypoxia-induced degradation of FTO promotes apoptosis by unmasking RACK1-mediated activation of MTK1-JNK1/2 pathway.

doi: 10.1016/j.jare.2025.01.019

Figure Lengend Snippet: Fig. 9. Verification of the anti-apoptotic role of the FTO-RACK1-MTK1-JNK1/2 cascade under hypoxia. A–D, NIH/3T3 cells were transfected with pcDNA 3.1, FTO-MYC (335– 502 aa), FTO-MYC (1–167 aa), or mtk1 siRNA or treated with 10 lM SP600125 for 12 h and cultured for an additional 12 h or 24 h under hypoxia (1 % O2). Cells exposed to hypoxia for 12 h were collected to measure the protein levels of RACK1, MYC, MTK1, JNK1/2, and cleaved Caspase-3 using Western blot analysis. Immunoprecipitation assays were conducted to analyze the binding of RACK1 with MYC, JNK1/2, or MTK1, as well as the interaction of MTK1 with JNK1/2 (A and B). Cells exposed to hypoxia for 24 h were collected to determine apoptotic rates using flow cytometric analysis (C), with the percentage of apoptotic cells shown in (D).

Article Snippet: Antibodies against RACK1 (27592–1-AP), NEDD4 (21698–1-AP), and MTK1 (21610–1-AP) were obtained from Proteintech.

Techniques: Transfection, Cell Culture, Western Blot, Immunoprecipitation, Binding Assay

Journal: Journal of advanced research

Article Title: Hypoxia-induced degradation of FTO promotes apoptosis by unmasking RACK1-mediated activation of MTK1-JNK1/2 pathway.

doi: 10.1016/j.jare.2025.01.019

Figure Lengend Snippet: Fig. 10. In vivo validation of the mechanism by which FTO degradation regulates apoptosis in porcine follicular granulosa cells. A–D, Granulosa cells were isolated from 20 follicles approximately 4 mm in diameter and classified into low and high hypoxia groups according to HIF-1a protein levels. Cell lysate was collected to measure the protein levels of FTO, ATG7, p62, MAP1LC3B, NEDD4, p-JNK1/2 (Thr183/Tyr185), JNK1/2, cleaved Caspase-3, RACK1, and MTK1 using Western blot analysis (A–D). Binding between FTO and NEDD4 (B), K48- and K63-linked ubiquitination levels of FTO protein (B), binding of RACK1 with FTO, MTK1, or JNK1/2 (D), and binding between MTK1 and JNK1/2 (D) were determined by immunoprecipitation assays. E and F, Granulosa cells were isolated from 15 follicles approximately 4 mm in diameter and divided into low and high groups according to FTO protein levels. The protein levels of p-JNK1/2 (Thr183/Tyr185), JNK1/2, cleaved Caspase-3, RACK1, and MTK1 were determined by Western blot (E). Binding of RACK1 and FTO, MTK1, or JNK1/2 and binding between MTK1 and JNK1/2 were analyzed by immunoprecipitation assays (F).

Article Snippet: Antibodies against RACK1 (27592–1-AP), NEDD4 (21698–1-AP), and MTK1 (21610–1-AP) were obtained from Proteintech.

Techniques: In Vivo, Biomarker Discovery, Isolation, Western Blot, Binding Assay, Ubiquitin Proteomics, Immunoprecipitation

Journal: Cell Death Discovery

Article Title: MEKK4-mediated Phosphorylation of HOXA10 at Threonine 362 facilitates embryo adhesion to the endometrial epithelium

doi: 10.1038/s41420-022-01203-1

Figure Lengend Snippet: A The MEKK4-interacting protein HOXA10 was screened with Python software based on data modeling and protein structure prediction. (Blue, MEKK4; bold blue, MEKK4 kinase domain; Yellow, HOXA10; bold yellow, HOXA10 homeobox). B , C Combination of exogenous Flag-IP or Myc-IP with Western blotting analysis of the interaction between HOXA10 and MEKK4 in 293 T cells co-transfected with Myc-HOXA10 and MEKK4-Flag for 48 h. D Combination of exogenous anti-MEKK4 endogenous immunoprecipitation with Western blotting analysis of the interaction between HOXA10 and MEKK4 in Ishikawa cell lysates. Mouse IgG was used as a negative control. E Cellular localization of endogenous MEKK4 and HOXA10 in Ishikawa cells. (Scale bar = 56 µm) F Cellular localization of GFP-MEKK4 and RFP-HOXA10 in Ishikawa cells. (Scale bar = 56 µm) G Combination of exogenous Flag-IP with Western blotting analysis of the interaction between HOXA10 and MEKK4 mutant constructs in 293 T cells. H Combination of exogenous Myc-IP with Western blotting analysis of the interaction between MEKK4 and HOXA10 mutant constructs in 293 T cells.

Article Snippet: Equal amounts of recombinant MEKK4 (Kinase activity, P5591, Abnova) were incubated with purified protein different fragments of HOXA10 in 25 μL kinase buffer (40 mM HEPES (pH 7.4), 20 mmol/L MgCl 2 , 1 mM DTT, and 10 μm ATP) for 60 min at 30 °C.

Techniques: Software, Western Blot, Transfection, Immunoprecipitation, Negative Control, Mutagenesis, Construct

Journal: Cell Death Discovery

Article Title: MEKK4-mediated Phosphorylation of HOXA10 at Threonine 362 facilitates embryo adhesion to the endometrial epithelium

doi: 10.1038/s41420-022-01203-1

Figure Lengend Snippet: A – D Immunohistochemical analysis of the MEKK4 expression in proliferative ( n = 6) and secretory ( n = 6) endometria from normal fertile women. Mouse IgG was used as a negative control. Data are represented as mean ± SEM, * P < 0.05, ** P < 0.01, using t -test. (Scale bar = 100 µm). E Western blotting analysis of MEKK4, HOXA10, and ITGB3 expression in Ishikawa cells treated with E 2 and MPA as indicated. F In vitro adhesion experiments analysis of the effect of MEKK4 knockdown on BeWo adhesion attached to the monolayer of Ishikawa cell. Values represent the mean ± SEM ( n = 3); * P < 0.05, using one-way ANOVA. G Western blotting analysis of MEKK4, HOXA10, ITGB3, FAK, and pho-FAK expression in Ishikawa cells treated with the adenovirus Ad-shMEKK4 and then stimulated with E 2 and MPA for 24 h. H In vitro adhesion experiments analysis of the effect of MEKK4 overexpression and kinase inactivation on BeWo adhesion attached to the monolayer of Ishikawa cell. Values represent the mean ± SEM ( n = 3); **** P < 0.0001; ** P < 0.01; * P < 0.05 compared with the pCDNA-Flag group; ## P < 0.01; # P < 0.05, using one-way ANOVA. I Western blotting analysis of MEKK4, HOXA10, ITGB3, FAK, and pho-FAK expression in Ishikawa cells transfected with MEKK4-Flag and MEKK4 mut-Flag (K1372R, kinase inactivation) and then treated with E 2 and MPA for 24 h.

Article Snippet: Equal amounts of recombinant MEKK4 (Kinase activity, P5591, Abnova) were incubated with purified protein different fragments of HOXA10 in 25 μL kinase buffer (40 mM HEPES (pH 7.4), 20 mmol/L MgCl 2 , 1 mM DTT, and 10 μm ATP) for 60 min at 30 °C.

Techniques: Immunohistochemical staining, Expressing, Negative Control, Western Blot, In Vitro, Knockdown, Over Expression, Transfection

Journal: Cell Death Discovery

Article Title: MEKK4-mediated Phosphorylation of HOXA10 at Threonine 362 facilitates embryo adhesion to the endometrial epithelium

doi: 10.1038/s41420-022-01203-1

Figure Lengend Snippet: A , B Real-time PCR analysis of MEKK4 and HOXA10 mRNA levels in Ishikawa cells infected with the adenovirus Ad-MEKK4-Flag for 48 h. Data are represented as mean ± SEM, *** P < 0.001, using one-way ANOVA. C Luciferase reporter assay analysis of the ITGB3-Luc activities of Ishikawa cells transfected with MEKK4-Flag, MEKK4 mut-Flag, Myc-HOXA10 (150 ng), and ITGB3-Luc (150 ng) for 48 h. Data are represented as mean ± SEM, **** P < 0.0001, *** P < 0.001, using one-way ANOVA. D HOXA10 mediated MEKK4-promoted BeWo adhesion. Values represent the mean ± SEM ( n = 3); ** P < 0.01; * P < 0.05, using one-way ANOVA. E MEKK4 promoted HOXA10-induced BeWo adhesion, depending on its kinase activity. Values represent the mean ± SEM ( n = 3); ** P < 0.01; * P < 0.05, using one-way ANOVA.

Article Snippet: Equal amounts of recombinant MEKK4 (Kinase activity, P5591, Abnova) were incubated with purified protein different fragments of HOXA10 in 25 μL kinase buffer (40 mM HEPES (pH 7.4), 20 mmol/L MgCl 2 , 1 mM DTT, and 10 μm ATP) for 60 min at 30 °C.

Techniques: Real-time Polymerase Chain Reaction, Infection, Luciferase, Reporter Assay, Transfection, Activity Assay

Journal: Cell Death Discovery

Article Title: MEKK4-mediated Phosphorylation of HOXA10 at Threonine 362 facilitates embryo adhesion to the endometrial epithelium

doi: 10.1038/s41420-022-01203-1

Figure Lengend Snippet: A Prokaryotic protein purification of various GST-HOXA10 mutant protein constructs (Coomassie blue staining). B MEKK4 phosphorylated various HOXA10 homeobox mutant constructs according to in vitro kinase assays. The solutions were analyzed by western blotting. C GST-HOXA10 319-410 was examined through mass spectrometry after SDS-PAGE electrophoresis by Thomas Brilliant Blue staining. D , E HOXA10 mutation did not affect the interaction between HOXA10 and MEKK4. F MEKK4 did not phosphorylate GST-HOXA10 319-410 T362A according to in vitro kinase assay. G 50 μg/ml CHX was added to the Ishikawa cells stimulated with Myc-HOXA10 T362A or Myc-HOXA10 WT for 2, 4, and 8 h. Western blot analysis of the levels of remaining HOXA10 in the cell extracts. The plots were relative to the levels at the 0 h time point. Values represent the mean ± SEM ( n = 2); * P < 0.05 versus HOXA10 WT alone, using one-way ANOVA. H Luciferase reporter assay analysis of the ITGB3-Luc activities of Ishikawa cells transfected with MEKK4-Flag (300 ng), Myc-HOXA10 WT (150 ng), Myc-HOXA10 T362A (150 ng), and ITGB3-Luc (150 ng) for 48 h. Values represent the mean ± SEM ( n = 3); **** P < 0.0001; *** P < 0.001, using one-way ANOVA. I After the HOXA10 mutation, the effect of MEKK4 on enhancing HOXA10-promoted BeWo adhesion was reduced. Values represent the mean ± SEM ( n = 3); * P < 0.05; *** P < 0.001, using one-way ANOVA. J Western blotting analysis of Flag, Myc, ITGB3, FAK, and pho-FAK expression in Ishikawa cells stimulated with MEKK4-Flag, Myc-HOXA10 WT , and Myc-HOXA10 T362A for 48 h.

Article Snippet: Equal amounts of recombinant MEKK4 (Kinase activity, P5591, Abnova) were incubated with purified protein different fragments of HOXA10 in 25 μL kinase buffer (40 mM HEPES (pH 7.4), 20 mmol/L MgCl 2 , 1 mM DTT, and 10 μm ATP) for 60 min at 30 °C.

Techniques: Protein Purification, Mutagenesis, Construct, Staining, In Vitro, Western Blot, Mass Spectrometry, SDS Page, Electrophoresis, Kinase Assay, Luciferase, Reporter Assay, Transfection, Expressing

Journal: Cell Death Discovery

Article Title: MEKK4-mediated Phosphorylation of HOXA10 at Threonine 362 facilitates embryo adhesion to the endometrial epithelium

doi: 10.1038/s41420-022-01203-1

Figure Lengend Snippet: A – C Immunohistochemical analysis of the MEKK4 and ITGB3 expression in the endometrial epithelia of fertile women ( n = 6) and RIF patients ( n = 6). The negative controls (NC) were mouse IgG and rabbit IgG. (Scale bar = 100 µm, Data are represented as mean ± SEM, ** P < 0.01, * P < 0.05, using t -test). D – F Western blotting and quantitative densitometry analysis for MEKK4 and ITGB3 in the endometria of RIF patients ( n = 20) and controls ( n = 20). Data are represented as mean ± SEM, **** P < 0.0001; ** P < 0.01, using t -test. G Correlation between MEKK4 and ITGB3 protein expression in the endometria of RIF patients and the FER group ( r = 0.63, **** P < 0.0001, using simple linear regression and Pearson correlation coefficient).

Article Snippet: Equal amounts of recombinant MEKK4 (Kinase activity, P5591, Abnova) were incubated with purified protein different fragments of HOXA10 in 25 μL kinase buffer (40 mM HEPES (pH 7.4), 20 mmol/L MgCl 2 , 1 mM DTT, and 10 μm ATP) for 60 min at 30 °C.

Techniques: Immunohistochemical staining, Expressing, Western Blot

Journal: Cell Death Discovery

Article Title: MEKK4-mediated Phosphorylation of HOXA10 at Threonine 362 facilitates embryo adhesion to the endometrial epithelium

doi: 10.1038/s41420-022-01203-1

Figure Lengend Snippet: During the window of implantation, MEKK4 expression was elevated under estrogen and progesterone treatment, and the kinase phosphorylated HOXA10, which in turn transcriptionally activated ITGB3 to promote embryo adhesion. This effect decreased when MEKK4 expression was reduced, which might be a novel mechanism for implantation failure in RIF patients.

Article Snippet: Equal amounts of recombinant MEKK4 (Kinase activity, P5591, Abnova) were incubated with purified protein different fragments of HOXA10 in 25 μL kinase buffer (40 mM HEPES (pH 7.4), 20 mmol/L MgCl 2 , 1 mM DTT, and 10 μm ATP) for 60 min at 30 °C.

Techniques: Expressing

Journal: Cell Death Discovery

Article Title: MEKK4-mediated Phosphorylation of HOXA10 at Threonine 362 facilitates embryo adhesion to the endometrial epithelium

doi: 10.1038/s41420-022-01203-1

Figure Lengend Snippet: Demographic details of the participants in the study of endometrial MEKK4, HOXA10 and ITGB3 expression.

Article Snippet: Equal amounts of recombinant MEKK4 (Kinase activity, P5591, Abnova) were incubated with purified protein different fragments of HOXA10 in 25 μL kinase buffer (40 mM HEPES (pH 7.4), 20 mmol/L MgCl 2 , 1 mM DTT, and 10 μm ATP) for 60 min at 30 °C.

Techniques: Expressing, Transplantation Assay

Journal: Cell Death Discovery

Article Title: MEKK4-mediated Phosphorylation of HOXA10 at Threonine 362 facilitates embryo adhesion to the endometrial epithelium

doi: 10.1038/s41420-022-01203-1

Figure Lengend Snippet: Primers.

Article Snippet: Equal amounts of recombinant MEKK4 (Kinase activity, P5591, Abnova) were incubated with purified protein different fragments of HOXA10 in 25 μL kinase buffer (40 mM HEPES (pH 7.4), 20 mmol/L MgCl 2 , 1 mM DTT, and 10 μm ATP) for 60 min at 30 °C.

Techniques: Sequencing, Plasmid Preparation, Construct

Journal: Neurobiology of disease

Article Title: Neuronal migration disorders : Focus on the cytoskeleton and epilepsy

doi: 10.1016/j.nbd.2015.08.003

Figure Lengend Snippet: Subcortical band heterotopia and periventricular heterotopia rodent models: Morphological phenotypes

Article Snippet: ; neurons − PVH − − − − Carabalona 2012 Mekk4 KO mouse Progen.

Techniques:

Journal: Neurobiology of disease

Article Title: Neuronal migration disorders : Focus on the cytoskeleton and epilepsy

doi: 10.1016/j.nbd.2015.08.003

Figure Lengend Snippet: Subcortical band heterotopia and periventricular heterotopia rodent models: Physiological and behavioral phenotypes

Article Snippet: ; neurons − PVH − − − − Carabalona 2012 Mekk4 KO mouse Progen.

Techniques: In Vitro