The levels of dCLK are substantially higher and hypo-phosphorylated at all times of the day in p{<i>dClk</i>-15A};<i>Clk</i>^out flies.

<p>(A–C) Adult flies of the indicated genotype were collected at different times of day (ZT), head extracts prepared and directly analyzed for immunoblotting (A) or processed for immunoprecipitation with anti-V5 Ab (B). β-Actin (ACTIN) served as a loading control. Immune complexes were further incubated in the absence (−) or presence (+) of λ phosphatase and immunoblotted with anti-V5 antibody, as indicated (B). Filled arrowheads denote hyper-phosphorylated isoforms of dCLK and open arrowhead denotes hypo-phosphorylated isoforms of dCLK (A). (C) Relative levels of dCLK were determined by measuring staining intensities using image J software. Shown are the average values from three independent experiments. (D) Total RNA was extracted from fly heads, and quantitative real-time RT-PCR was performed to measure the relative levels of <i>dClk</i> transcripts. Shown are the average values from three independent experiments using p{<i>dClk</i>-15A}, 6M;<i>Clk</i>^out flies. Error bars denote SEM.