antibodies mekk4 sc-100396  (Santa Cruz Biotechnology)

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: A non-translational role of threonyl-tRNA synthetase in regulating JNK signaling during myogenic differentiation

doi: 10.1096/fj.202101094R

Figure Lengend Snippet: MEKK4 mediates ThrRS regulation of JNK and myogenesis. (A-B) C2C12 myoblasts were transduced with lentiviruses expressing shMEKK4 or shScramble and selected by puromycin, followed by differentiation for 3 days. Protein expression and phosphorylation levels were assessed by western blotting (A) and differentiation was quantified by fusion index (B) (n = 3). Molecular weight markers in kDa are indicated on western blots. (C) C2C12 myoblasts were transduced with lentiviruses expressing shThrRS, with or without shMEKK4 and selected by puromycin, followed by differentiation for 3 days. Differentiation was quantified by fusion index. The change in fusion index upon ThrRS knockdown was compared between with and without shMEKK4 (right graph) (n = 3). All quantitative data are presented as mean ± SEM. Two-tailed paired t test was performed to compare data to control (shScramble) in A, B, and right graph of C. For data in C (left graph), two-way ANOVA analysis was performed, followed by Student–Newman–Keuls post hoc test for multiple comparisons. *p < .05, **p < .01. N.S: not significant

Article Snippet: Anti-Flag-BioM2 (F9291), anti-MEKK4 (M7194), and gelatin were purchased from Sigma.

Techniques: Transduction, Expressing, Western Blot, Molecular Weight, Two Tailed Test

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: A non-translational role of threonyl-tRNA synthetase in regulating JNK signaling during myogenic differentiation

doi: 10.1096/fj.202101094R

Figure Lengend Snippet: ThrRS regulates MEKK4 by interacting with Axin1. (A) HEK293T cells were transfected to express Flag-ThrRS or Flag-Axin1with empty vector as control. Flag-ThrRS or Flag-Axin1 was pulled down by anti-Flag M2 affinity gel followed by western blotting of endogenous Axin1 or ThrRS, respectively (n = 3). (B) Cells were transfected with Flag-tagged WT or fragments of ThrRS with empty vector as control. IP was performed with anti-Flag M2 affinity gel, followed by western blotting of endogenous Axin1 (n = 3). (C) Cells were transfected to express Flag-Axin1 with or without HA-MEKK4. IP was performed with anti-HA-agarose followed by western blotting (n = 3). (D) Cells were transfected to express Flag-Axin1 with or without HA-MEKK4 or Flag-ThrRS. IP was performed with anti-HA-agarose followed by western blotting (n = 3). (E) Cells were transduced with lentiviruses expressing shThrRS or shScramble and then transfected to express Flag-Axin1 and HA-MEKK4. IP was performed with anti-Flag M2 affinity gel followed by western blotting (n = 3) (F) Cells were transfected to express Flag-ThrRS with or without Flag-Axin1 for 24 h, followed by cell lysis and western blot analysis of pJNK (n = 3). (G) A proposed model of ThrRS regulation of myogenesis. Molecular weight markers in kDa are indicated on all western blots. In E and F, quantitative data are presented as mean ± SEM. Two-tailed paired t test was performed to compare the data as indicated. **p < .01. N.S: not significant

Article Snippet: Anti-Flag-BioM2 (F9291), anti-MEKK4 (M7194), and gelatin were purchased from Sigma.

Techniques: Transfection, Plasmid Preparation, Western Blot, Transduction, Expressing, Lysis, Molecular Weight, Two Tailed Test

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: 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: The membranes were then blocked with Tris-buffered saline solution containing 5% nonfat milk for 1 h, and further incubated with the following primary antibodies: MEKK4 (1:500; sc-100396, Santa Cruz), ITGB3 (1:1000; BS3660, Bioworld), pho-Ser (1:1 000; P3430, Sigma), pho-Thr (1:1 000; PT-5H5, Thermo), FAK (1:1 000; 12636-1-AP Abways), p-FAK (Y397) (1:1 000; 10357-1-AP, Abways), and GAPDH (1:10 000; AP0063, Bioworld), which served as an internal control.

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: The membranes were then blocked with Tris-buffered saline solution containing 5% nonfat milk for 1 h, and further incubated with the following primary antibodies: MEKK4 (1:500; sc-100396, Santa Cruz), ITGB3 (1:1000; BS3660, Bioworld), pho-Ser (1:1 000; P3430, Sigma), pho-Thr (1:1 000; PT-5H5, Thermo), FAK (1:1 000; 12636-1-AP Abways), p-FAK (Y397) (1:1 000; 10357-1-AP, Abways), and GAPDH (1:10 000; AP0063, Bioworld), which served as an internal control.

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: The membranes were then blocked with Tris-buffered saline solution containing 5% nonfat milk for 1 h, and further incubated with the following primary antibodies: MEKK4 (1:500; sc-100396, Santa Cruz), ITGB3 (1:1000; BS3660, Bioworld), pho-Ser (1:1 000; P3430, Sigma), pho-Thr (1:1 000; PT-5H5, Thermo), FAK (1:1 000; 12636-1-AP Abways), p-FAK (Y397) (1:1 000; 10357-1-AP, Abways), and GAPDH (1:10 000; AP0063, Bioworld), which served as an internal control.

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: The membranes were then blocked with Tris-buffered saline solution containing 5% nonfat milk for 1 h, and further incubated with the following primary antibodies: MEKK4 (1:500; sc-100396, Santa Cruz), ITGB3 (1:1000; BS3660, Bioworld), pho-Ser (1:1 000; P3430, Sigma), pho-Thr (1:1 000; PT-5H5, Thermo), FAK (1:1 000; 12636-1-AP Abways), p-FAK (Y397) (1:1 000; 10357-1-AP, Abways), and GAPDH (1:10 000; AP0063, Bioworld), which served as an internal control.

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: The membranes were then blocked with Tris-buffered saline solution containing 5% nonfat milk for 1 h, and further incubated with the following primary antibodies: MEKK4 (1:500; sc-100396, Santa Cruz), ITGB3 (1:1000; BS3660, Bioworld), pho-Ser (1:1 000; P3430, Sigma), pho-Thr (1:1 000; PT-5H5, Thermo), FAK (1:1 000; 12636-1-AP Abways), p-FAK (Y397) (1:1 000; 10357-1-AP, Abways), and GAPDH (1:10 000; AP0063, Bioworld), which served as an internal control.

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: The membranes were then blocked with Tris-buffered saline solution containing 5% nonfat milk for 1 h, and further incubated with the following primary antibodies: MEKK4 (1:500; sc-100396, Santa Cruz), ITGB3 (1:1000; BS3660, Bioworld), pho-Ser (1:1 000; P3430, Sigma), pho-Thr (1:1 000; PT-5H5, Thermo), FAK (1:1 000; 12636-1-AP Abways), p-FAK (Y397) (1:1 000; 10357-1-AP, Abways), and GAPDH (1:10 000; AP0063, Bioworld), which served as an internal control.

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: The membranes were then blocked with Tris-buffered saline solution containing 5% nonfat milk for 1 h, and further incubated with the following primary antibodies: MEKK4 (1:500; sc-100396, Santa Cruz), ITGB3 (1:1000; BS3660, Bioworld), pho-Ser (1:1 000; P3430, Sigma), pho-Thr (1:1 000; PT-5H5, Thermo), FAK (1:1 000; 12636-1-AP Abways), p-FAK (Y397) (1:1 000; 10357-1-AP, Abways), and GAPDH (1:10 000; AP0063, Bioworld), which served as an internal control.

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: The membranes were then blocked with Tris-buffered saline solution containing 5% nonfat milk for 1 h, and further incubated with the following primary antibodies: MEKK4 (1:500; sc-100396, Santa Cruz), ITGB3 (1:1000; BS3660, Bioworld), pho-Ser (1:1 000; P3430, Sigma), pho-Thr (1:1 000; PT-5H5, Thermo), FAK (1:1 000; 12636-1-AP Abways), p-FAK (Y397) (1:1 000; 10357-1-AP, Abways), and GAPDH (1:10 000; AP0063, Bioworld), which served as an internal control.

Techniques: Sequencing, Plasmid Preparation, 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 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: The cells were hatched with an anti-HOXA10 antibody (1: 200; Boster) and an anti-MEKK4 antibody (1: 200; Santa Cruz) at 4 °C overnight.

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: The cells were hatched with an anti-HOXA10 antibody (1: 200; Boster) and an anti-MEKK4 antibody (1: 200; Santa Cruz) at 4 °C overnight.

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: The cells were hatched with an anti-HOXA10 antibody (1: 200; Boster) and an anti-MEKK4 antibody (1: 200; Santa Cruz) at 4 °C overnight.

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: The cells were hatched with an anti-HOXA10 antibody (1: 200; Boster) and an anti-MEKK4 antibody (1: 200; Santa Cruz) at 4 °C overnight.

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: The cells were hatched with an anti-HOXA10 antibody (1: 200; Boster) and an anti-MEKK4 antibody (1: 200; Santa Cruz) at 4 °C overnight.

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: The cells were hatched with an anti-HOXA10 antibody (1: 200; Boster) and an anti-MEKK4 antibody (1: 200; Santa Cruz) at 4 °C overnight.

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: The cells were hatched with an anti-HOXA10 antibody (1: 200; Boster) and an anti-MEKK4 antibody (1: 200; Santa Cruz) at 4 °C overnight.

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: The cells were hatched with an anti-HOXA10 antibody (1: 200; Boster) and an anti-MEKK4 antibody (1: 200; Santa Cruz) at 4 °C overnight.

Techniques: Sequencing, Plasmid Preparation, Construct