Journal: PLoS Biology
Article Title: The CK2 Kinase Stabilizes CLOCK and Represses Its Activity in the Drosophila Circadian Oscillator
Figure Lengend Snippet: PER, TIM, and CLK are found in protein complexes containing CK2. Anti-FLAG immunoprecipitation (IP) from nonsonicated head extracts of flies collected at DD1. FLAG-CK2α or FLAG-CK2β was immunoprecipitated from 1 mg of total protein extracts from heads and 50% of the precipitate was subjected to Western blot analysis. We loaded 50 µg of head extracts as input controls. IgG LC indicates the immunoglobulin G light chain used for the precipitation that is well detected by the anti-mouse secondary antibody. Gray and black bars represent subjective day and subjective night, respectively. Experiments were performed at least twice. (A, Left) FLAG-CK2α was immunoprecipitated from tim > FLAG-CkIIα flies and tim-gal4 /+ negative controls (control) in a per + background. IP of CK2α (FLAG) and co-IP of CK2β, TIM, PER, and CLK were visualized by immunoblotting. «*» shows an aspecific band recognized by the anti-PER antibody. (Right) TIM, PER, and CLK immunoblots of the corresponding inputs on per + background. The time “CT10” indicates a mixed population of flies harvested at CT8 and CT12. A CB stained band in the size range of CLK is used as a loading control. TIM was run on a 3–8% Tris-Acetate gel. (B, Left) FLAG-CK2β was immunoprecipitated from tim > FLAG-CkIIß flies and tim-gal4 /+ negative controls (control) in a per + background. IP of CK2ß (with anti-CK2ß) and co-IP of CK2α, TIM, PER, and CLK were visualized by immunoblotting. (Right) TIM, PER, and CLK immunoblots of the corresponding inputs. A CB stained band in the size range of CLK is used as a loading control. (C) FLAG-CK2α or FLAG-CK2β was immunoprecipitated from tim > FLAG-CkIIα ( α ) flies and tim-gal4 /+ negative controls (C) in a per 0 background. co-IP of CLK was visualized by immunoblotting. Input samples and immunoprecipitates were run on the same gel for C and α. (D) Image showing PDF (green), CK2α (magenta), and CLK (blue) fluorescent immunolabeling in small PDF + LNv-s of an adult fly at ZT3. The fourth square is a composite picture of the three stainings. Single optical planes are shown taken by confocal microscopy.
Article Snippet: Samples (50 µg) for FLAG, CK2α, and CK2β immunoblots were run on NuPAGE Novex 4–12% Bis-Tris precast gels (Life Technologies).
Techniques: Immunoprecipitation, Western Blot, Co-Immunoprecipitation Assay, Staining, Immunolabeling, Confocal Microscopy