Fig. 4. HLH-2 binds to both E-boxes in the ACEL. (A) Synthesis and purification of FLAG-Luciferase and FLAG-HLH-2 proteins. Both proteins were expressed in insect cells using a baculovirus expression system and then purified using Ni²-columns. The purified proteins were resolved on an SDS-PAGE gel for Coomassie staining and immunoblotting with anti-FLAG antibodies. (B) Probe A contains both E-boxes, probes B and C contain only one E-box, and probe D does not contain any E-boxes. The probes contain an extra 70 bp at the 3' end of the ACEL. Thus, the first E-box (E-1) is near the 5' end of the probe and the second E-box (E-2) is in the middle of the probe. (C) An EMSA that shows the binding of the HLH-2 protein to both E-boxes in the ACEL. The purified proteins were incubated with the wild-type and the mutated DNA probes of the ACEL (Fig. 3B). F indicates the migration of free DNA probe, B is the FLAG-HLH-2 protein/DNA probe complex, and Supershift is created by adding anti-FLAG antibodies (lanes 6 to 8).