1. SLK/LOK-phosphorylated and activated ezrin prevents MISP localization at the cell cortex.
SLK/LOK-phosphorylated and activated ezrin prevents MISP localization at the cell cortex. (A) HeLa cells were treated with either control, ezrin_1 siRNA or with a combination of ezrin_1 siRNA and different GFP–ezrin siRNA-resistant constructs. Representative images show staining of MISP (magenta) and ezrin or the GFP signal (green); DNA was co-stained with Hoechst (blue). The cortical-to-cytoplasmic MISP ratio in control siRNA-transfected cells was normalized to 1.0. Data represent mean±s.d. from at least three independent experiments, n>45 cells. (B) HeLa cells were treated for 6 h with either DMSO or the ezrin inhibitor NSC (10 µM). The ratio of cortical-to-cytoplasmic MISP levels was quantified relative to the DMSO-treated cells (set at 1.0). Data represent mean±s.d. from three independent experiments, n>59 cells. Representative images show staining with MISP (green) and Hoechst (DNA, blue). (C) HeLa cells were transfected with indicated siRNAs, and the cortical-to-cytoplasmic MISP levels quantified, with the control treated cells normalized to 1.0. Data represent mean±s.d. from three independent experiments, n>53 cells. Representative immunofluorescence images of HeLa cells stained with anti-MISP antibody (green) and co-stained with Hoechst (DNA, blue). (D) HeLa cells were treated with control or ezrin_2 siRNA for 72 h. Before harvesting, cells were treated for 10 min with DMSO (control) or Latrunculin B (LatrB). The cortical-to-cytoplasmic MISP ratio was quantified and normalized to the control. Data represent mean±s.d. from three independent experiments, n>50 cells. (E) Actin co-sedimentation assay with actin, GST–MISP and His–ezrin. Supernatant (S) and pellet (P) samples were analyzed by western blotting using indicated antibodies. The signal intensity ratio of pelleted MISP to pelleted actin or pelleted ezrin to pelleted actin was quantified and normalized to the sample in which the respective protein was solely added to actin. *P<0.05, ****P< (unpaired Student's t-test).
Yvonne T. Kschonsak, and Ingrid Hoffmann J Cell Sci 2018;131:jcs214544
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