CLK GP47 antiserum cross-reacts with DAC. (A) Western blots containing 1.0 μl and 5.0 μl of in vitro transcribed and translated DAC (IVTT DAC), 0.1 μl and 1.0 u μl of in vitro transcribed and translated CLK (IVTT CLK), and 100 μg of head extract from flies collected at ZT14 probed with either DAC monoclonal antibody (DAC mAb) (left) or CLK GP47 antiserum (GP47) (right). (B) Western blots of samples as denoted in panel A probed with CLK GP47 and 10 μg of purified DAC (GP47 + DAC) (left) or CLK GP47 alone (GP47) (right). (C) Dissected CNSs from wild-type L3 larvae collected at ZT21 were incubated with CLK GP47 antiserum, PER antiserum, DAC monoclonal antibody and 10 μg of purified DAC antigen. A 32 μm projected Z-series image of CLK GP47 IR alone (W.T. CLK), PER IR alone (W.T. PER), DAC IR alone (W.T. DAC), and merged CLK GP47, PER and DAC IR (W.T. merged). Arrows denote brain oscillator cells. s-LNv, small ventral lateral neurons; DN2, dorsal neuron 2 s; DN1*, dorsal neuron 1 s plus additional brain oscillator neurons that may include DN3s, l-LNvs and LNds.