Journal: Cells

Article Title: Protective Effects of SIRT6 Overexpression against DSS-Induced Colitis in Mice

doi: 10.3390/cells9061513

Figure Lengend Snippet: SIRT6 regulated expression and phosphorylation of TAK1. ( a ) The expression and phosphorylation levels of TAK1 were determined by immunohistochemistry. ( b , c ) The statistical results of ( a ). n = 3–6, ** p < 0.01.

Article Snippet: Sections then were blocked in 5% rat serum and incubated overnight at 4 °C with the diluted biotinylated primary antibody (1:500; rat-anti-mouse; anti-TAK1, #ab109526, Abcam, Cambridge, England, UK; anti-p-TAK1, #4508, Cell Signaling Technology, Danvers, MA, USA; anti-c-Jun, #ab40766, Abcam; anti-p-Jun, #ab32385, Abcam; anti-NF-κB p65 antibody, #ab32536, Abcam; anti-NF-κB p65 (phosphor S536), #ab86299, Abcam; anti-NF-κB p65 (acetyl K310) antibody, #ab19870, Abcam).

Techniques: Expressing, Immunohistochemistry

Journal: eLife

Article Title: TAK1-mediated phosphorylation of PLCE1 represses PIP2 hydrolysis to impede esophageal squamous cancer metastasis

doi: 10.7554/eLife.97373

Figure Lengend Snippet: ( A ) Increased expression of TAK1 in ECA-109 cells transfected with a plasmid expressing Map3k7 . ( B ) Increased expression of TAK1 inhibits the morphological changes to form spindle-shaped mesenchymal cells induced by epidermal growth factor (EGF) (100 ng/ml) in ECA-109. Scale bar = 100 µm. ( C ) Increased expression of TAK1 inhibits cell migration and invasion in ECA-109 cells. Cell migration and invasion were analyzed by transwell assay. n=4 biologically independent replicates. ( D ) Wound healing assay showing cell migration was attenuated by TAK1. n=5 biologically independent replicates. ( E ) TAK1 decreased mesenchymal marker gene expression, while increased the expression of epithelial markers. ECA-109 cells were transfected with the plasmid carrying Map3k7. 24 hr post-transfection, protein samples were prepared and subjected to western blot. Actin was used as a loading control. ( F ) Knockdown of TAK1 increased the expression of F-Actin. ECA-109 cells were transfected with Map3k7 siRNA. 72 hr post-transfection, cells were subjected to immunofluorescence analysis using an anti-F-Actin antibody (red). Hoechst was used to stain the nucleus (blue). Scale bar = 10 µm. ( G ) TAK1 knockdown induces spindle-shaped mesenchymal cell morphology in ECA-109 cells. Scale bar = 100 µm. ( H ) Reduced expression of TAK1 promotes cell migration and invasion. ECA-109 cells were transfected with Map3k7 siRNA. 72 hr post-transfection, cell migration and invasion were analyzed by transwell assay. n=5 biologically independent replicates. ( I ) Knockdown of TAK1 increases mesenchymal protein marker expression and decreases epithelial protein marker expression. Data are presented as mean ± SD. Statistical significance was tested by unpaired Student’s t-test. *p<0.05, **p<0.01, and ***p<0.001. Figure 1—source data 1. TAK1 negatively regulates esophageal squamous cell carcinoma (ESCC) migration and invasion. Figure 1—source data 2. PDF file containing original western blots for , indicating the relevant bands. Figure 1—source data 3. Original files for western blot analysis displayed in .

Article Snippet: Rabbit monoclonal antibodies against TAK1 (#5206), phospho-TAK1 (Ser412, #9339), PKCα (#2056), phospho-PKC (pan) (gamma Thr514) (#38938), GSK-3β (#9315), phospho-GSK-3β (Ser9) (#5558), β-Catenin (#8480), phospho-β-Catenin (Ser33/37/Thr41) (#9561), MMP-2 (#87809), mouse monoclonal antibodies against Actin (#3700), Myc-Tag (Sepharose Bead Conjugate) (#55464), Epithelial-Mesenchymal Transition (EMT) antibody sample kit (#9782), phosphor-IKK (#2078), IKK (#61294), phosphor-JNK (#4668), JNK (#9252), phospho-ERK (#4370), ERK (#9102), phospho-P38 MAPK (#9211), P38 MPAK (#9212), and normal rabbit IgG (#2729) were from Cell Signaling Technology (Beverley, MA, USA).

Techniques: Expressing, Transfection, Plasmid Preparation, Migration, Transwell Assay, Wound Healing Assay, Marker, Gene Expression, Western Blot, Control, Knockdown, Immunofluorescence, Staining

Journal: eLife

Article Title: TAK1-mediated phosphorylation of PLCE1 represses PIP2 hydrolysis to impede esophageal squamous cancer metastasis

doi: 10.7554/eLife.97373

Figure Lengend Snippet: Cells were transfected with a plasmid expressing Map3k7. 24 or 48 hr post-transfection, cells were subjected to transwell ( A ) and wound healing assay ( B ). Scale bar = 500 µm ( A ) and 100 µm ( B ). ( C ) Quantified protein expression based on western blot data in . n=3 biologically independent replicates. ( D ) TAK1 decreased mesenchymal marker gene expression, while increased the expression of epithelial markers. Gene expression was analyzed by quantitative real time-PCR (qRT-PCR), and Gapdh was used as a house-keeping gene. n=4 biologically independent replicates. Data are presented as mean ± SD. Statistical significance was tested by unpaired Student’s t-test. *p<0.05, **p<0.01, and ***p<0.001 . Figure 1—figure supplement 1—source data 1. TAK1 represses cell migration in ECA-109 cells.

Article Snippet: Rabbit monoclonal antibodies against TAK1 (#5206), phospho-TAK1 (Ser412, #9339), PKCα (#2056), phospho-PKC (pan) (gamma Thr514) (#38938), GSK-3β (#9315), phospho-GSK-3β (Ser9) (#5558), β-Catenin (#8480), phospho-β-Catenin (Ser33/37/Thr41) (#9561), MMP-2 (#87809), mouse monoclonal antibodies against Actin (#3700), Myc-Tag (Sepharose Bead Conjugate) (#55464), Epithelial-Mesenchymal Transition (EMT) antibody sample kit (#9782), phosphor-IKK (#2078), IKK (#61294), phosphor-JNK (#4668), JNK (#9252), phospho-ERK (#4370), ERK (#9102), phospho-P38 MAPK (#9211), P38 MPAK (#9212), and normal rabbit IgG (#2729) were from Cell Signaling Technology (Beverley, MA, USA).

Techniques: Transfection, Plasmid Preparation, Expressing, Wound Healing Assay, Western Blot, Marker, Gene Expression, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Migration

Journal: eLife

Article Title: TAK1-mediated phosphorylation of PLCE1 represses PIP2 hydrolysis to impede esophageal squamous cancer metastasis

doi: 10.7554/eLife.97373

Figure Lengend Snippet: ( A–C ) Knockdown of TAK1 in ECA-109 cells promotes cell migration and invasion analyzed by transwell ( A ) and wound healing ( B ) assays. Scale bar = 500 µm ( A ) or 100 µm ( B ). ( C ) Quantitative analysis of the western blot data as shown in . n=3 biologically independent replicates. ( D ) Reduced expression of TAK1 in ECA-109 cells increases mesenchymal marker gene expression, while decreases epithelial marker gene expression. Gene expression was analyzed by quantitative real time-PCR (qRT-PCR), and Gapdh was used as a house-keeping gene. n=4 biologically independent replicates. Data are presented as mean ± SD. Statistical significance was tested by unpaired Student’s t-test. *p<0.05, **p<0.01, and ***p<0.001 . Figure 1—figure supplement 2—source data 1. TAK1 silencing promotes esophageal squamous cell carcinoma (ESCC) migration and invasion.

Article Snippet: Rabbit monoclonal antibodies against TAK1 (#5206), phospho-TAK1 (Ser412, #9339), PKCα (#2056), phospho-PKC (pan) (gamma Thr514) (#38938), GSK-3β (#9315), phospho-GSK-3β (Ser9) (#5558), β-Catenin (#8480), phospho-β-Catenin (Ser33/37/Thr41) (#9561), MMP-2 (#87809), mouse monoclonal antibodies against Actin (#3700), Myc-Tag (Sepharose Bead Conjugate) (#55464), Epithelial-Mesenchymal Transition (EMT) antibody sample kit (#9782), phosphor-IKK (#2078), IKK (#61294), phosphor-JNK (#4668), JNK (#9252), phospho-ERK (#4370), ERK (#9102), phospho-P38 MAPK (#9211), P38 MPAK (#9212), and normal rabbit IgG (#2729) were from Cell Signaling Technology (Beverley, MA, USA).

Techniques: Knockdown, Migration, Western Blot, Expressing, Marker, Gene Expression, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

Journal: eLife

Article Title: TAK1-mediated phosphorylation of PLCE1 represses PIP2 hydrolysis to impede esophageal squamous cancer metastasis

doi: 10.7554/eLife.97373

Figure Lengend Snippet: ( A–B ) Knockdown of TAK1 by Map3k7 shRNA. ECA-109 cells were transduced with lentivirus bearing Map3k7 shRNA (LV- Map3k7 shRNA) or NC shRNA (LV-NC). 48 hr post-transduction, cells were harvested for analyzing TAK1 expression by quantitative real time-PCR (qRT-PCR) ( A ) and western blot ( B ). n = 3 biologically independent replicates. ( C–E ) Decreased expression of TAK1 facilitates cell migration and invasion. ECA-109 cells were transduced with LV- Map3k7 shRNA or LV-NC. 48 hr post-transduction, cells were subjected to transwell ( C, D ) or wound healing ( E ) assay. n=5 biologically independent replicates. Scale bar = 500 µm ( C ); scale bar = 100 µm ( E ). ( F – G ) Reduced expression of TAK1 increases mesenchymal marker expression and decreases epithelial marker expression. n=3 biologically independent replicates. ( H ) Reduced expression of TAK1 in ECA-109 cells affects epithelial-mesenchymal transition (EMT) related gene expression. Protein levels were analyzed by western blot, and Actin was used as a loading control. Gene expression was detected by qRT-PCR, and Gapdh was used as a house-keeping gene. n=4 biologically independent replicates. Data are presented as mean ± SD. Statistical significance was tested by unpaired Student’s t-test. *p<0.05, **p<0.01, and ***p<0.001. Figure 1—figure supplement 3—source data 1. TAK1 knockdown facilitates esophageal squamous cell carcinoma (ESCC) migration and invasion. Figure 1—figure supplement 3—source data 2. PDF file containing original western blots for , indicating the relevant bands. Figure 1—figure supplement 3—source data 3. Original files for western blot analysis displayed in .

Article Snippet: Rabbit monoclonal antibodies against TAK1 (#5206), phospho-TAK1 (Ser412, #9339), PKCα (#2056), phospho-PKC (pan) (gamma Thr514) (#38938), GSK-3β (#9315), phospho-GSK-3β (Ser9) (#5558), β-Catenin (#8480), phospho-β-Catenin (Ser33/37/Thr41) (#9561), MMP-2 (#87809), mouse monoclonal antibodies against Actin (#3700), Myc-Tag (Sepharose Bead Conjugate) (#55464), Epithelial-Mesenchymal Transition (EMT) antibody sample kit (#9782), phosphor-IKK (#2078), IKK (#61294), phosphor-JNK (#4668), JNK (#9252), phospho-ERK (#4370), ERK (#9102), phospho-P38 MAPK (#9211), P38 MPAK (#9212), and normal rabbit IgG (#2729) were from Cell Signaling Technology (Beverley, MA, USA).

Techniques: Knockdown, shRNA, Transduction, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Migration, Marker, Gene Expression, Control

Journal: eLife

Article Title: TAK1-mediated phosphorylation of PLCE1 represses PIP2 hydrolysis to impede esophageal squamous cancer metastasis

doi: 10.7554/eLife.97373

Figure Lengend Snippet: ( A–B ) TAK1 expression was decreased by Map3k7 gRNA in ECA-109 cells. TAK1 knockout was achieved by CRISPR-Cas9. (B–D) Knockout of TAK1 expression in ECA-109 cells accelerates cell migration and invasion as analyzed by transwell ( B, C ) and wound healing ( D ) assays. Scale bar = 500 µm ( B ); scale bar = 100 µm ( D ). ( E – F ) Loss of TAK1 increases mesenchymal protein marker expression and reduces epithelial protein marker expression. ECA-109 cells were treated with Map3k7 gRNA, and then cells were harvested for western blot analysis. n=3 biologically independent replicates. ( G ) Knockout of TAK1 expression in ECA-109 cells alters epithelial-mesenchymal transition (EMT) related gene expression as analyzed by quantitative real time-PCR (qRT-PCR). Gapdh was used as a house-keeping gene. n=4 biologically independent replicates. Protein levels were analyzed by western blot, and Actin was used as a loading control. Data are presented as mean ± SD. Statistical significance was tested by unpaired Student’s t-test. *p<0.05, **p<0.01, and ***p<0.001. Figure 1—figure supplement 4—source data 1. TAK1 knockout accelerates esophageal squamous cell carcinoma (ESCC) migration and invasion. Figure 1—figure supplement 4—source data 2. PDF file containing original western blots for , indicating the relevant bands. Figure 1—figure supplement 4—source data 3. Original files for western blot analysis displayed in .

Article Snippet: Rabbit monoclonal antibodies against TAK1 (#5206), phospho-TAK1 (Ser412, #9339), PKCα (#2056), phospho-PKC (pan) (gamma Thr514) (#38938), GSK-3β (#9315), phospho-GSK-3β (Ser9) (#5558), β-Catenin (#8480), phospho-β-Catenin (Ser33/37/Thr41) (#9561), MMP-2 (#87809), mouse monoclonal antibodies against Actin (#3700), Myc-Tag (Sepharose Bead Conjugate) (#55464), Epithelial-Mesenchymal Transition (EMT) antibody sample kit (#9782), phosphor-IKK (#2078), IKK (#61294), phosphor-JNK (#4668), JNK (#9252), phospho-ERK (#4370), ERK (#9102), phospho-P38 MAPK (#9211), P38 MPAK (#9212), and normal rabbit IgG (#2729) were from Cell Signaling Technology (Beverley, MA, USA).

Techniques: Expressing, Knock-Out, CRISPR, Migration, Marker, Western Blot, Gene Expression, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Control

Journal: eLife

Article Title: TAK1-mediated phosphorylation of PLCE1 represses PIP2 hydrolysis to impede esophageal squamous cancer metastasis

doi: 10.7554/eLife.97373

Figure Lengend Snippet: Cells were treated with (5Z)-7-oxozeaenol (Oxo; 10 µM), or NG25 (10 µM), or Takinib (10 µM) for 24 hr, and then cell morphology, migration, and invasion were analyzed. ( A ) Inhibition of TAK1 promotes morphological changes to form spindle-shaped mesenchymal cells in ECA-109 cells. Scale bar = 100 µm. ( B–E ) Inhibition of TAK1 promotes cell migration and invasion in ECA-109 cells. Cell migration and invasion were analyzed by transwell ( B, C ) and wound healing ( D, E ) assays. Scale bar = 500 µm ( B ); scale bar = 100 µm ( D ). n=5 biologically independent replicates. Data are presented as mean ± SD. Statistical significance was tested by unpaired Student’s t-test. * p<0.05 and *** p < 0.001 . Figure 1—figure supplement 5—source data 1. Inhibition of TAK1 potentiates cell migration and invasion in ECA-109 cells.

Article Snippet: Rabbit monoclonal antibodies against TAK1 (#5206), phospho-TAK1 (Ser412, #9339), PKCα (#2056), phospho-PKC (pan) (gamma Thr514) (#38938), GSK-3β (#9315), phospho-GSK-3β (Ser9) (#5558), β-Catenin (#8480), phospho-β-Catenin (Ser33/37/Thr41) (#9561), MMP-2 (#87809), mouse monoclonal antibodies against Actin (#3700), Myc-Tag (Sepharose Bead Conjugate) (#55464), Epithelial-Mesenchymal Transition (EMT) antibody sample kit (#9782), phosphor-IKK (#2078), IKK (#61294), phosphor-JNK (#4668), JNK (#9252), phospho-ERK (#4370), ERK (#9102), phospho-P38 MAPK (#9211), P38 MPAK (#9212), and normal rabbit IgG (#2729) were from Cell Signaling Technology (Beverley, MA, USA).

Techniques: Migration, Inhibition

Journal: eLife

Article Title: TAK1-mediated phosphorylation of PLCE1 represses PIP2 hydrolysis to impede esophageal squamous cancer metastasis

doi: 10.7554/eLife.97373

Figure Lengend Snippet: ( A ) Tandem mass spectrometry showing serine 1060 (S1060) in PLCE1 was phosphorylated by TAK1. ECA-109 cells were transfected with a plasmid expressing Map3k7 . 24 hr post-transfection, cells were harvested and subjected to co-immunoprecipitation. The resulting immunocomplex was analyzed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). ( B ) TAK1 fails to phosphorylate PLCE1 S1060A . ECA-109 cells were co-transfected with the plasmids carrying wildtype (WT) Plce1 , mutated Plce1 (PLCE1 S1060A ), or Map3k7 as indicated. 24 hr post-transfection, cells were collected for western blot analysis. ( C–E ) Inhibition of TAK1 reduces PLCE1 phosphorylation at S1060. ECA-109 cells were co-transfected with the plasmids expressing Plce1 or Map3k7 . 6 hr post-transfection, TAK1 inhibitor (5Z)-7-oxozeaenol (Oxo; 10 µM) ( C ), or 10 µM Takinib ( D ), or 10 µM NG25 ( E ) was added in culture medium, and cells were cultured for additional 18 hr. Cells were then subjected to western blot analysis. Actin was used as a loading control. ( F–G ) Immunohistochemical analysis of TAK1 ( F ) and p-PLCE1 ( G ) expression in normal and esophageal squamous tumor tissues. n=4 biologically independent replicates. Scale bar = 20 µm. ( H ) Correlation between p-PLCE1 and TAK1 based on immunohistochemical data as shown in ( F–G ). 10 views for each sample were randomly chosen for Pearson’s correlation test. ( I ) TAK1 and p-PLCE1 protein levels in clinical samples. Protein levels were analyzed by western blot, and Actin was used as a loading control. n=4 biologically independent replicates. N: normal tissue; T: tumor tissue. Figure 2—source data 1. TAK1 phosphorylates PLCE1 at serine 1060. Figure 2—source data 2. PDF file containing original western blots for , indicating the relevant bands. Figure 2—source data 3. Original files for western blot analysis displayed in .

Article Snippet: Rabbit monoclonal antibodies against TAK1 (#5206), phospho-TAK1 (Ser412, #9339), PKCα (#2056), phospho-PKC (pan) (gamma Thr514) (#38938), GSK-3β (#9315), phospho-GSK-3β (Ser9) (#5558), β-Catenin (#8480), phospho-β-Catenin (Ser33/37/Thr41) (#9561), MMP-2 (#87809), mouse monoclonal antibodies against Actin (#3700), Myc-Tag (Sepharose Bead Conjugate) (#55464), Epithelial-Mesenchymal Transition (EMT) antibody sample kit (#9782), phosphor-IKK (#2078), IKK (#61294), phosphor-JNK (#4668), JNK (#9252), phospho-ERK (#4370), ERK (#9102), phospho-P38 MAPK (#9211), P38 MPAK (#9212), and normal rabbit IgG (#2729) were from Cell Signaling Technology (Beverley, MA, USA).

Techniques: Mass Spectrometry, Transfection, Plasmid Preparation, Expressing, Immunoprecipitation, Liquid Chromatography, Liquid Chromatography with Mass Spectroscopy, Western Blot, Inhibition, Phospho-proteomics, Cell Culture, Control, Immunohistochemical staining

Journal: eLife

Article Title: TAK1-mediated phosphorylation of PLCE1 represses PIP2 hydrolysis to impede esophageal squamous cancer metastasis

doi: 10.7554/eLife.97373

Figure Lengend Snippet: ( A ) TAK1 overexpression induces endogenous PLCE1 phosphorylation at serine 1060 (p-PLCE1 S1060). ECA-109 cells were transfected with a plasmid expressing TAK1. 24 hr post-transfection, cells were harvested and subjected to western blot analysis. Actin was used as a loading control. ( B ) TAK1 knockdown reduces endogenous p-PLCE1 S1060. ECA-109 cells were transfected with Map3k7 siRNA. 48 hr post-transfection, cells were collected for analyzing p-PLCE1 S1060. Actin was used as a loading control. ( C ) TAK1 phosphorylates PLCE1 at S1060 in an in vitro kinase assay. ECA-109 cells were transfected with a plasmid carrying S protein tagged TAK1 (SP-TAK1), Myc tagged PLCE1 (Myc-PLCE1), or Myc tagged PLCE1 S1060A (Myc-PLCE1 S1060A). 24 hr post-transfection, cells were harvested and subjected to protein pull-down, the resulting purified SP-TAK1, Myc-PLCE1, and Myc-PLCE1 S1060A were incubated in a kinase assay buffer. The reaction mixtures were then resolved by SDS-PAGE, the resulting gels were analyzed by Coomassie blue staining or western blot. Lane M: marker; Lane 1: SP-TAK1+Myc-PLCE1; Lane 2: SP-TAK1+Myc-PLCE1 S1060A. The observed band for p-PLCE1 in lane 2 is likely due to the presence of endogenous wildtype PLCE1 in the TAK1 pull-down samples. Figure 2—figure supplement 1—source data 1. PDF file containing original western blots for , indicating the relevant bands. Figure 2—figure supplement 1—source data 2. Original files for western blot analysis displayed in .

Article Snippet: Rabbit monoclonal antibodies against TAK1 (#5206), phospho-TAK1 (Ser412, #9339), PKCα (#2056), phospho-PKC (pan) (gamma Thr514) (#38938), GSK-3β (#9315), phospho-GSK-3β (Ser9) (#5558), β-Catenin (#8480), phospho-β-Catenin (Ser33/37/Thr41) (#9561), MMP-2 (#87809), mouse monoclonal antibodies against Actin (#3700), Myc-Tag (Sepharose Bead Conjugate) (#55464), Epithelial-Mesenchymal Transition (EMT) antibody sample kit (#9782), phosphor-IKK (#2078), IKK (#61294), phosphor-JNK (#4668), JNK (#9252), phospho-ERK (#4370), ERK (#9102), phospho-P38 MAPK (#9211), P38 MPAK (#9212), and normal rabbit IgG (#2729) were from Cell Signaling Technology (Beverley, MA, USA).

Techniques: Over Expression, Phospho-proteomics, Transfection, Plasmid Preparation, Expressing, Western Blot, Control, Knockdown, In Vitro, Kinase Assay, Purification, Incubation, SDS Page, Staining, Marker

Journal: eLife

Article Title: TAK1-mediated phosphorylation of PLCE1 represses PIP2 hydrolysis to impede esophageal squamous cancer metastasis

doi: 10.7554/eLife.97373

Figure Lengend Snippet: ( A–B ) Effects of TAK1 on PLCE1 ( A ) and PLCE1 S1060A ( B ) enzyme activity. ECA-109 cells were co-transfected with the plasmids expressing Plce1-Myc or Map3k7 . 24 hr post-transfection, cells were subjected to pull-down assay by using the beads with anti-Myc antibody. PLCE1 enzyme activity was assayed by Phospholipases C (PLC) Activity Assay Kit. n=3 biologically independent replicates. ( C–E ) TAK1 abolishes PLCE1-induced inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) in ECA-109 ( C ), KYSE-150 ( D ), and TE-1 cells ( E ). Cells were transfected with the plasmids bearing Plce1 or Map3k7 as indicated. 24 hr post-transfection, cells were harvested for measuring IP3 and DAG. n=3 biologically independent replicates. ( F ) TAK1 attenuates PLCE1-induced intracellular Ca 2+ ([Ca 2+ ]). ECA-109 cells were transfected with the plasmids bearing Plce1 or Map3k7 as indicated. [Ca 2+ ] was labeled with Fluo-4 AM, which was then detected by a fluorescent microscope. Scale bar = 20 µm. ( G ) Quantified fluorescence intensity of [Ca 2+ ] in ECA-109 cells. n=3 biologically independent replicates. ( H ) Fluorescence intensity of Fluo-4 in ECA-109, KYSE-150, and TE-1 cells was examined with a fluorospectrophotometer. n=3 biologically independent replicates. ( I ) Flow cytometry analysis of [Ca 2+ ]. Cell treatments were described in ( F ). Data are presented as mean ± SD. Statistical significance was tested by unpaired Student’s t-test ( A–B ) or two-tailed one-way ANOVA test ( C–E, G–I ). * p < 0.05, ** p < 0.01, and *** p<0.001 . Figure 4—source data 1. TAK1 inhibits PLCE1 enzyme activity.

Article Snippet: Rabbit monoclonal antibodies against TAK1 (#5206), phospho-TAK1 (Ser412, #9339), PKCα (#2056), phospho-PKC (pan) (gamma Thr514) (#38938), GSK-3β (#9315), phospho-GSK-3β (Ser9) (#5558), β-Catenin (#8480), phospho-β-Catenin (Ser33/37/Thr41) (#9561), MMP-2 (#87809), mouse monoclonal antibodies against Actin (#3700), Myc-Tag (Sepharose Bead Conjugate) (#55464), Epithelial-Mesenchymal Transition (EMT) antibody sample kit (#9782), phosphor-IKK (#2078), IKK (#61294), phosphor-JNK (#4668), JNK (#9252), phospho-ERK (#4370), ERK (#9102), phospho-P38 MAPK (#9211), P38 MPAK (#9212), and normal rabbit IgG (#2729) were from Cell Signaling Technology (Beverley, MA, USA).

Techniques: Activity Assay, Transfection, Expressing, Pull Down Assay, Labeling, Microscopy, Fluorescence, Flow Cytometry, Two Tailed Test

Journal: eLife

Article Title: TAK1-mediated phosphorylation of PLCE1 represses PIP2 hydrolysis to impede esophageal squamous cancer metastasis

doi: 10.7554/eLife.97373

Figure Lengend Snippet: ECA-109 cells were transfected with plasmids expressing Plce1 , mutated Plce1 (S1060A), or Map3k7 as indicated. 36 hr post-transfection, cell migration and invasion were assayed by transwell assay ( A–B ) and wound healing assay ( C–D ). n=5 biologically independent replicates. Scale bar = 500 µm ( A ); scale bar = 100 µm ( C ). Data are presented as mean ± SD. Statistical significance was tested by two-tailed one-way ANOVA test. *** p<0.001. Figure 4—figure supplement 1—source data 1. The negative impact of TAK1 on PLCE1-induced cell migration and invasion requires TAK1 kinase activity.

Article Snippet: Rabbit monoclonal antibodies against TAK1 (#5206), phospho-TAK1 (Ser412, #9339), PKCα (#2056), phospho-PKC (pan) (gamma Thr514) (#38938), GSK-3β (#9315), phospho-GSK-3β (Ser9) (#5558), β-Catenin (#8480), phospho-β-Catenin (Ser33/37/Thr41) (#9561), MMP-2 (#87809), mouse monoclonal antibodies against Actin (#3700), Myc-Tag (Sepharose Bead Conjugate) (#55464), Epithelial-Mesenchymal Transition (EMT) antibody sample kit (#9782), phosphor-IKK (#2078), IKK (#61294), phosphor-JNK (#4668), JNK (#9252), phospho-ERK (#4370), ERK (#9102), phospho-P38 MAPK (#9211), P38 MPAK (#9212), and normal rabbit IgG (#2729) were from Cell Signaling Technology (Beverley, MA, USA).

Techniques: Transfection, Expressing, Migration, Transwell Assay, Wound Healing Assay, Two Tailed Test, Activity Assay

Journal: eLife

Article Title: TAK1-mediated phosphorylation of PLCE1 represses PIP2 hydrolysis to impede esophageal squamous cancer metastasis

doi: 10.7554/eLife.97373

Figure Lengend Snippet: ( A ) TAK1 overexpression activates p-IKK, p-JNK, p-ERK, p-P38 MAPK. ECA-109 cells were transfected with a plasmid expressing TAK1. 24 hr post-transfection, cells were collected for western blot analysis. ( B ) Quantification data for the blots in ( A ). ( C ) TAK1 knockdown represses p-IKK, p-JNK, p-ERK, p-P38 MAPK. ECA-109 cells were transfected with Map3k7 siRNA. 48 hr post-transfection, cells were harvested for western blot analysis. ( D ) Quantification data for the blots in ( C ). EV: empty vector; NC: negative control. Actin was used a loading control. *p < 0.05, **p < 0.01, and ***p < 0.001, by unpaired Student’s t-test. Figure 4—figure supplement 2—source data 1. Effects of TAK1 on p-IKK, p-JNK, p-ERK, and p-P38 MAPK in ECA-109 cells. Figure 4—figure supplement 2—source data 2. PDF file containing original western blots for , indicating the relevant bands. Figure 4—figure supplement 2—source data 3. Original files for western blot analysis displayed in .

Article Snippet: Rabbit monoclonal antibodies against TAK1 (#5206), phospho-TAK1 (Ser412, #9339), PKCα (#2056), phospho-PKC (pan) (gamma Thr514) (#38938), GSK-3β (#9315), phospho-GSK-3β (Ser9) (#5558), β-Catenin (#8480), phospho-β-Catenin (Ser33/37/Thr41) (#9561), MMP-2 (#87809), mouse monoclonal antibodies against Actin (#3700), Myc-Tag (Sepharose Bead Conjugate) (#55464), Epithelial-Mesenchymal Transition (EMT) antibody sample kit (#9782), phosphor-IKK (#2078), IKK (#61294), phosphor-JNK (#4668), JNK (#9252), phospho-ERK (#4370), ERK (#9102), phospho-P38 MAPK (#9211), P38 MPAK (#9212), and normal rabbit IgG (#2729) were from Cell Signaling Technology (Beverley, MA, USA).

Techniques: Over Expression, Transfection, Plasmid Preparation, Expressing, Western Blot, Knockdown, Negative Control, Control

Journal: eLife

Article Title: TAK1-mediated phosphorylation of PLCE1 represses PIP2 hydrolysis to impede esophageal squamous cancer metastasis

doi: 10.7554/eLife.97373

Figure Lengend Snippet: ( A ) IP3R blocking inhibits PLCE1-induced signal transduction in the axis of PKC/GSK-3β/β-Catenin. ECA-109 cells were transfected with the plasmid expressing Plce1 for 6 hr and then treated with 2-APB (10 µM) for additional 18 hr. ( B ) [Ca 2+ ] blocking represses signal transduction in the axis of PKC/GSK-3β/β-Catenin induced by PLCE1. ECA-109 cells were transfected with the plasmid expressing Plce1 for 6 hr and then treated with BAPTA-AM (10 µM) for additional 18 hr. ( C ) PKC inhibition blocks PLCE1 stimulated signal transduction in the axis of PKC/GSK-3β/β-Catenin. ECA-109 cells were transfected with the plasmid expressing Plce1 . 6 hr post-transfection, cells were treated with 100 nM of Midostaurin for additional 18 hr. ( D ) PKC inhibition represses PLCE1-induced nuclear translocation of β-Catenin in ECA-109 cells. Cells were transfected with the plasmid expressing PLCE1. 6 hr post-transfection, 2-APB (10 µM), BAPTA-AM (10 µM), or Midostaurin (100 nM) was added in culture medium, and cells were cultured for additional 18 hr. Scale bar = 10 µm. Immunofluorescence was used to examine subcellular distribution of β-Catenin. ( E ) TAK1 counteracts PLCE1-induced signal transduction in the axis of PKC/GSK-3β/β-Catenin. ECA-109 cells were transfected with the plasmids expressing Plce1 or Map3k7 as indicated for 24 hr. ( F ) TAK1 reduces PLCE1-induced nuclear distribution of β-Catenin in ECA-109 cells. Cells were transfected with the plasmids expressing Plce1 or Map3k7 as indicated. Scale bar = 10 µm. ( G ) Dominant negative TAK1 (K63W) fails to block signal transduction in the axis of PKC/GSK-3β/β-Catenin/MMP2 induced by PLCE1. ECA-109 cells were transfected with the plasmids expressing Plce1 or mutated Map3k7 (TAK1 K63W) for 24 hr. ( H ) TAK1 has no effect on PLCE1 S1060A-induced signal transduction in the axis of PKC/GSK-3β/β-Catenin. ECA-109 cells were transfected with the plasmids expressing PLCE1 S1060A or TAK1 for 24 hr. Protein levels were analyzed by western blot, and Actin was used as a loading control. Representative blots were shown. Figure 5—source data 1. PDF file containing original western blots for , indicating the relevant bands. Figure 5—source data 2. Original files for western blot analysis displayed in .

Article Snippet: Rabbit monoclonal antibodies against TAK1 (#5206), phospho-TAK1 (Ser412, #9339), PKCα (#2056), phospho-PKC (pan) (gamma Thr514) (#38938), GSK-3β (#9315), phospho-GSK-3β (Ser9) (#5558), β-Catenin (#8480), phospho-β-Catenin (Ser33/37/Thr41) (#9561), MMP-2 (#87809), mouse monoclonal antibodies against Actin (#3700), Myc-Tag (Sepharose Bead Conjugate) (#55464), Epithelial-Mesenchymal Transition (EMT) antibody sample kit (#9782), phosphor-IKK (#2078), IKK (#61294), phosphor-JNK (#4668), JNK (#9252), phospho-ERK (#4370), ERK (#9102), phospho-P38 MAPK (#9211), P38 MPAK (#9212), and normal rabbit IgG (#2729) were from Cell Signaling Technology (Beverley, MA, USA).

Techniques: Blocking Assay, Transduction, Transfection, Plasmid Preparation, Expressing, Inhibition, Translocation Assay, Cell Culture, Immunofluorescence, Dominant Negative Mutation, Western Blot, Control

Journal: eLife

Article Title: TAK1-mediated phosphorylation of PLCE1 represses PIP2 hydrolysis to impede esophageal squamous cancer metastasis

doi: 10.7554/eLife.97373

Figure Lengend Snippet: ( A–F ) Quantified data for the western blots as shown in . n=3 biologically independent replicates. Data are presented as mean ± SD. Statistical significance was tested by two-tailed one-way ANOVA test. *p<0.05, **p<0.01, and ***p<0.001. Figure 5—figure supplement 7—source data 1. TAK1 mitigates PLCE1-induced signal transduction in the axis of PKC/GSK-3β/β-Catenin.

Article Snippet: Rabbit monoclonal antibodies against TAK1 (#5206), phospho-TAK1 (Ser412, #9339), PKCα (#2056), phospho-PKC (pan) (gamma Thr514) (#38938), GSK-3β (#9315), phospho-GSK-3β (Ser9) (#5558), β-Catenin (#8480), phospho-β-Catenin (Ser33/37/Thr41) (#9561), MMP-2 (#87809), mouse monoclonal antibodies against Actin (#3700), Myc-Tag (Sepharose Bead Conjugate) (#55464), Epithelial-Mesenchymal Transition (EMT) antibody sample kit (#9782), phosphor-IKK (#2078), IKK (#61294), phosphor-JNK (#4668), JNK (#9252), phospho-ERK (#4370), ERK (#9102), phospho-P38 MAPK (#9211), P38 MPAK (#9212), and normal rabbit IgG (#2729) were from Cell Signaling Technology (Beverley, MA, USA).

Techniques: Western Blot, Two Tailed Test, Transduction

Journal: eLife

Article Title: TAK1-mediated phosphorylation of PLCE1 represses PIP2 hydrolysis to impede esophageal squamous cancer metastasis

doi: 10.7554/eLife.97373

Figure Lengend Snippet: Each mouse was intravenously injected with 1×10 6 ECA-109 cells diluted in 100 µl PBS. Mice were treated with Takinib at the dosage of 50 mg/kg/day for 15 days, mice in control group were received vehicle (corn oil). Eight weeks later, mice were sacrificed, and the lungs and livers from each group were collected and photographed. ( A ) Typical images of specimens. ( B ) Hematoxylin and eosin staining of metastatic nodules in lungs. ( C ) The number of nodules in lungs. ( D ) Takinib treatment induces signal transduction in the axis of PKC/GSK-3β/β-Catenin. Protein levels were analyzed by western blot, and Actin was used as a loading control. n=3 biologically independent replicates. ( E ) Quantitative analysis of the western blot data shown in ( D ). Data are presented as mean ± SD. Statistical significance was tested by unpaired Student’s t-test. * p < 0.05 , ** p<0.01, and *** p<0.001 . Figure 6—source data 1. Inhibition of TAK1 by Takinib promotes esophageal squamous cell carcinoma (ESCC) metastasis in nude mice. Figure 6—source data 2. PDF file containing original western blots for , indicating the relevant bands. Figure 6—source data 3. Original files for western blot analysis displayed in .

Article Snippet: Rabbit monoclonal antibodies against TAK1 (#5206), phospho-TAK1 (Ser412, #9339), PKCα (#2056), phospho-PKC (pan) (gamma Thr514) (#38938), GSK-3β (#9315), phospho-GSK-3β (Ser9) (#5558), β-Catenin (#8480), phospho-β-Catenin (Ser33/37/Thr41) (#9561), MMP-2 (#87809), mouse monoclonal antibodies against Actin (#3700), Myc-Tag (Sepharose Bead Conjugate) (#55464), Epithelial-Mesenchymal Transition (EMT) antibody sample kit (#9782), phosphor-IKK (#2078), IKK (#61294), phosphor-JNK (#4668), JNK (#9252), phospho-ERK (#4370), ERK (#9102), phospho-P38 MAPK (#9211), P38 MPAK (#9212), and normal rabbit IgG (#2729) were from Cell Signaling Technology (Beverley, MA, USA).

Techniques: Injection, Control, Staining, Transduction, Western Blot, Inhibition

Journal: eLife

Article Title: TAK1-mediated phosphorylation of PLCE1 represses PIP2 hydrolysis to impede esophageal squamous cancer metastasis

doi: 10.7554/eLife.97373

Figure Lengend Snippet:

Article Snippet: Rabbit monoclonal antibodies against TAK1 (#5206), phospho-TAK1 (Ser412, #9339), PKCα (#2056), phospho-PKC (pan) (gamma Thr514) (#38938), GSK-3β (#9315), phospho-GSK-3β (Ser9) (#5558), β-Catenin (#8480), phospho-β-Catenin (Ser33/37/Thr41) (#9561), MMP-2 (#87809), mouse monoclonal antibodies against Actin (#3700), Myc-Tag (Sepharose Bead Conjugate) (#55464), Epithelial-Mesenchymal Transition (EMT) antibody sample kit (#9782), phosphor-IKK (#2078), IKK (#61294), phosphor-JNK (#4668), JNK (#9252), phospho-ERK (#4370), ERK (#9102), phospho-P38 MAPK (#9211), P38 MPAK (#9212), and normal rabbit IgG (#2729) were from Cell Signaling Technology (Beverley, MA, USA).

Techniques: Transfection, Construct, shRNA, Recombinant, Plasmid Preparation, Sequencing, Activity Assay, Enzyme-linked Immunosorbent Assay, Software

Journal: Nature Communications

Article Title: Oligomerization-primed coiled-coil domain interaction with Ubc13 confers processivity to TRAF6 ubiquitin ligase activity

doi: 10.1038/s41467-017-01290-0

Figure Lengend Snippet: Only long free polyubiquitin chains potently activate TAK1 kinase. a Purification scheme of polyubiquitin (polyUb) chains. b Purification scheme of endogenous TAK1 kinase complex. c Potent TAK1 activation by high molecular size polyUb chains. Partially purified TAK1 complex as in b was incubated with different sizes of polyUb chains after their fractionation as in a in the presence of ATP for 60 min at 30 °C. Aliquots of the reaction products were analyzed by immunoblotting with the indicated antibodies. The size distribution of the polyUb chains is shown in the bottom panel. d The relative amounts of polyUb chains used in c were determined by immunoblotting with anti-Ub antibody after their complete disassembly by DUB vOTU. e The chain length rather than the concentration of polyUb chains contributes more to TAK1 stimulatory activity. As in c , TAK1 was incubated with increasing amounts of long polyUb chains (Fraction A6) or short polyUb chains (Fraction B7) to determine its dose response to polyUb chains. S.E. short exposure, L.E. long exposure, M.W. molecular weight (KDa)

Article Snippet: Mouse antibody against ubiquitin (sc-8017, dilution 1:1000) was purchased from Santa Cruz Biotechnology; Rabbit antibodies against p-TAK1 Thr187 (#4536, dilution 1:1000), p-p38 Thr180/Tyr182 (#9211, dilution 1:1000), p-JNK Thr183/Tyr185 (#4668, dilution 1:1000), p38 (#8690, dilution 1:1000), JNK (#9252, dilution 1:1000), p-ERK1/2 (#4370, dilution 1:1000), and K63-linkage-specific polyubiquitin chains (#5621, dilution 1:1000) were purchased from Cell Signaling; Mouse monoclonal antibody against p-IκBα Ser32/36 (#9246, dilution 1:1000) was purchased from Cell Signaling.

Techniques: Purification, Activation Assay, Incubation, Fractionation, Western Blot, Concentration Assay, Activity Assay, Molecular Weight

Journal: Nature Communications

Article Title: Oligomerization-primed coiled-coil domain interaction with Ubc13 confers processivity to TRAF6 ubiquitin ligase activity

doi: 10.1038/s41467-017-01290-0

Figure Lengend Snippet: CC domain mediates TRAF6 intermolecular interaction and oligomerization. a Amino acid sequence alignment of TRAF6 CC domains from different species. The heptad repeats are denoted with “a-b-c-d-e-f-g”. Arrows indicate the mutation residues used in the study. b CC domain mediates TRAF6 self-association. Flag-tagged TRAF6, mutant M3, or T6RZ were co-transfected with Myc-tagged TRAF6 or with Myc-tagged EGFP into HEK293T cells and subjected to immunoprecipitation with anti-Myc antibodies. The immunoprecipitated complexes (upper panels) and cell lysates (down panels) were immunoblotted with the indicated antibodies. c CC domain mediates TRAF6 oligomerization. TRAF6-Flag and TRAF6-M3-Flag were expressed in HEK 293T cells and purified using anti-Flag M2 magnetic beads. The eluted proteins were analyzed by 10% native gel. d CC domain mediates TRAF6 oligomerization. T6RZC and T6RZC-M3 were purified from Sf9 cells and separated by size-exclusion column Superdex 200. Aliquots from each fraction were separated by 10% SDS-PAGE and detected by Coomassie blue staining. Superimposed gel filtration profiles of T6RZC and T6RZC-M3 are shown below. e , f Disruption of CC oligomerization impairs polyUb chain synthesis. Time-course analysis of polyUb chain synthesis was performed to compare T6RZC and T6RZC-M3. Time-dependent accumulation of polyUb chains was shown in e and quantified in f . R ob , arbitrary observed rate of polyUb assembly. g Only the high molecular size TRAF6 can activate TAK1 and IKK. Aliquots from d were subjected to in vitro IKK activation assay. Activation of TAK1 and IKK was determined with the indicated antibodies

Article Snippet: Mouse antibody against ubiquitin (sc-8017, dilution 1:1000) was purchased from Santa Cruz Biotechnology; Rabbit antibodies against p-TAK1 Thr187 (#4536, dilution 1:1000), p-p38 Thr180/Tyr182 (#9211, dilution 1:1000), p-JNK Thr183/Tyr185 (#4668, dilution 1:1000), p38 (#8690, dilution 1:1000), JNK (#9252, dilution 1:1000), p-ERK1/2 (#4370, dilution 1:1000), and K63-linkage-specific polyubiquitin chains (#5621, dilution 1:1000) were purchased from Cell Signaling; Mouse monoclonal antibody against p-IκBα Ser32/36 (#9246, dilution 1:1000) was purchased from Cell Signaling.

Techniques: Sequencing, Mutagenesis, Transfection, Immunoprecipitation, Purification, Magnetic Beads, SDS Page, Staining, Filtration, Disruption, In Vitro, Activation Assay

Journal: Nature Communications

Article Title: Oligomerization-primed coiled-coil domain interaction with Ubc13 confers processivity to TRAF6 ubiquitin ligase activity

doi: 10.1038/s41467-017-01290-0

Figure Lengend Snippet: CC domain-mediated oligomerization and interaction with Ubc13 are critical for the biological functions of TRAF6. a TRAF6 mutants M3, M4, and M5 fail to rescue the activation of TAK1, IKK, and MAPK kinases in the IL-1R signaling pathway. TRAF6 KO MEF cells reconstituted with TRAF6 WT or M3, M4, M5 mutants were stimulated with IL-1β for the indicated duration of time. Cell lysates were analyzed by immunoblotting with the indicated antibodies. To capture K63-linked polyubiquitin chains, cell lysates were subjected to GST-PAP80-UIMs-based pull down. Bound polyUb chains were separated on 6–18% SDS-PAGE and immunoblotted with an anti-K63 Ub antibody. b TRAF6 mutants M3, M4, and M5 fail to rescue the activation of NF-κB. As in a , but cells were untreated or treated with IL-1β for 3 and 6 h, respectively. Total RNAs were extracted, and qRT-PCR was performed to measure the induction of IL-6 and A20. The data are showed as the mean ± s.d. of triplicate independent sets of experiments. c Model depicting the contribution of CC domains to TRAF6 processivity. Upon receiving upstream activation signals, CC domains start to trimerize, which, on the one hand, leads to TRAF6 trimerization and effective RING domain trimerization and their binding to Ub~Ubc13/Uev2; on the other hand, trimerized CC domains themselves also bind to Ub~Ubc13/Uev2. These two events together contribute to processive polyUb chain assembly

Article Snippet: Mouse antibody against ubiquitin (sc-8017, dilution 1:1000) was purchased from Santa Cruz Biotechnology; Rabbit antibodies against p-TAK1 Thr187 (#4536, dilution 1:1000), p-p38 Thr180/Tyr182 (#9211, dilution 1:1000), p-JNK Thr183/Tyr185 (#4668, dilution 1:1000), p38 (#8690, dilution 1:1000), JNK (#9252, dilution 1:1000), p-ERK1/2 (#4370, dilution 1:1000), and K63-linkage-specific polyubiquitin chains (#5621, dilution 1:1000) were purchased from Cell Signaling; Mouse monoclonal antibody against p-IκBα Ser32/36 (#9246, dilution 1:1000) was purchased from Cell Signaling.

Techniques: Activation Assay, Western Blot, SDS Page, Quantitative RT-PCR, Binding Assay