Journal:

Article Title: ?-Arrestin1 modulates lymphoid enhancer factor transcriptional activity through interaction with phosphorylated dishevelled proteins

doi: 10.1073/pnas.211572798

Figure Lengend Snippet: Enhanced binding of βarr1 to phosphorylated Dvl. (A) HEK-293 cells were transfected with Myc-Dvl2 and FLAG-βarr1, and cell extracts were immunoprecipitated with anti-Myc antibody directed against Dvl2. Immunoprecipitated Dvl2 was phosphorylated in the presence of [γ-32P]ATP by endogenous Dvl2-associated kinase(s). Immunoprecipitated Dvl2 was processed for autoradiography (Upper) or incubated with His-βarr1 for >3 h at 4°C. Washed Dvl2 immunoprecipitates were analyzed by immunoblot with anti-His antibody to detect associated βarr1 (Lower). (B) MBP-Dvl1 was phosphorylated by protein kinase CKII in the presence of ATP. Phosphorylated and unphosphorylated control MBP-Dvl1 was incubated with His-βarr1 for >3 h at 4°C, and associated βarr1 was detected by immunoblot with anti-His antibody directed against βarr1. (C) MBP-Dvl1 was phosphorylated to various stoichiometric levels by protein kinase CKII. Phosphorylated MBP-Dvl1 was then either eluted in sample buffer and processed for autoradiography (Inset Upper) or incubated with His-βarr1. MBP-Dvl1-associated βarr1 was detected by immunoblot with anti-His antibody directed against βarr1 (Inset Lower). The relative amounts of βarr1 bound to MBP-Dvl1 phosphorylated by protein kinase CKII were quantified as fold increases over that bound to unphosphorylated MBP-Dvl1. Results are the mean ± SEM of three independent experiments.

Article Snippet: MBP-Dvl1 was washed with protein kinase CKII buffer (20 mM Tris, pH 7.5/50 mM KCl/2 mM MgCl 2 ) and phosphorylated with protein kinase CKII (New England Biolabs) and [γ- 32 P]ATP (60 μM; final concentration, 400 cpm/pmol) at 30°C for 30 min. Phosphorylated MBP-Dvl1 was visualized by exposure of dried gels to film.

Techniques: Binding Assay, Transfection, Immunoprecipitation, Autoradiography, Incubation, Western Blot