Fig. 1. Identification of the PER2 domains and residues required for PER2-CK1δ interaction.

a–c Western blot analyses of immunoprecipitation assays of FLAG-tagged PER constructs and GFP-tagged CK1δ were expressed in HEK293 cells (3 μg of PER plasmid and 2 μg of CK1δ plasmid transfected per 60-mm dish of cells) and showed that CK1δ associates with hPER when co-expressed in HEK293 cells. The levels of PER2 proteins and CK1δ were determined by western blot using anti-FLAG and GFP antibody, respectively. Analyses of the interaction of FLAG-tagged PER1, PER2, and PER3 with GFP-tagged CK1δ (a). Identification of the region of hPER2 sufficient for the hPER2-CK1δ interaction (b). Analyses of hPER2 deletion constructs (c). Three independent experiments were performed to validate the results. See also Fig. S1a–c. d Amino acid sequence alignment of PCD domains of hPER1-3 proteins. Yellow regions mark conserved residues in the α helical domain (top). Diagrams showing the secondary structure prediction of the hPER proteins (bottom). e Western blot analyses of immunoprecipitation assays of FLAG-tagged PER constructs with the indicated mutation and GFP-tagged CK1δ were expressed in HEK293 cells. Three independent experiments were performed to validate the results. f Structure prediction of the wild-type hPER2 (top right) and hPER2 (V729G-L730G) (bottom right) region described in 1d by Alphafold. The L730 residue in the hPER2 was indicated by a red arrow in an enlarged picture on the left. g Amino acid sequence alignment of the PCD domains of nine indicated vertebrate PER2 homologs.