Journal:

Article Title: Nucleotide Exchange Factor ECT2 Interacts with the Polarity Protein Complex Par6/Par3/Protein Kinase C? (PKC?) and Regulates PKC? Activity

doi: 10.1128/MCB.24.15.6665-6675.2004

Figure Lengend Snippet: Par6 and ECT2 activate Cdc42 and PKCζ. (A) (Left panel) HeLa cells transfected with Myc-ECT2-ΔN5 together with Flag-Par6C vector or vector alone were lysed, and GTP-bound Cdc42 was pulled down with GST-PBD beads. The Western blot was carried out with anti-Cdc42 antibody. (Right panel) Similar experiments were performed in MDCK cells except that Flag-ECT2-ΔN5 was used. Accumulation of GTP-bound Rac1 was also estimated (lower panel). Similar results were obtained in three independent experiments. (B) Silencing of ECT2 reduces GTP-bound Cdc42. HeLa cells were treated with ECT2 siRNA or control luciferase siRNA (co) for 5 h and then transfected with Flag-Par6C. Cells were lysed 20 h after transfection, and GTP-bound Cdc42 was pulled down with GST-PBD-Sepharose beads. Western blots were carried out with anti-Cdc42, anti-ECT2, or anti-Flag antibody. These results were reproduced in three independent experiments. (C) ECT2 stimulates PKCζ activity. Cos7 cells transfected with the HA-tagged PKCζ expression vector in combination with the ECT2 and Flag-Par6C vectors or vector alone were lysed, and HA-PKCζ was immunoprecipitated with anti-HA antibody. PKCζ activity was estimated by in vitro kinase assays with myelin basic protein as the substrate, followed by autoradiography (upper panel). The level of HA-PKCζ was detected by immunoblotting with anti-HA antibody (lower panel). X-ray-films were scanned, and signals were quantitated with NIH Image software. Data are representative of three independent experiments.

Article Snippet: GTP-bound Cdc42 was estimated by the Cdc42 activation kit (Cytoskeleton Inc.).

Techniques: Transfection, Plasmid Preparation, Western Blot, Luciferase, Activity Assay, Expressing, Immunoprecipitation, In Vitro, Autoradiography, Software