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
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![( A ) IP3R blocking inhibits PLCE1-induced signal transduction in the axis of <t>PKC/GSK-3β/β-Catenin.</t> 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 <t>)</t> <t>TAK1</t> 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 .](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_7773/pmc12017773/pmc12017773__elife-97373-fig5.jpg)
