Arnt and Miz-1 interact. A) Schematic representation of motifs within native and mutant constructs of Arnt. The human Arnt used in this study consists of 774 amino acids and the location of its helix-loop-helix (bHLH), Per-Arnt-Sim (PAS) and the glutamine rich (Q-rich) domains are shown. The amino acid mutations in “2xmut” and “4xmut” are indicated in bold in the Helix II sequence. B) Miz-1/GFP and wt or mutated variants of Arnt/Flag were overexpressed as indicated. Immunoprecipitation (IP) was performed using anti-GFP and immunoblotting (IB) was performed with anti-Flag (upper panel). Input (4%) was run on a parallel gel and IB was performed with anti-Flag (middle panel). In order to verify Miz-1/GFP IP in the different samples, the co-IP membrane was stripped and incubated with anti-GFP (lower panel). C) HEK293 cells were co-transfected with Miz-1/Flag and Arnt/CFP. Cells were subjected to confocal microscopy to visualize Texas-red fluorescence (for Miz-1/Flag; left panel) or cyan fluorescence (for Arnt/CFP; middle panel). The right panel shows the merged image. The borders of the cytoplasm of the cells are marked in white, while the borders of nuclei are marked in yellow.