PER is a substrate of CK1α.

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PER is a substrate of CK1α. PER is a substrate of CK1α. A, B, Drosophila S2 cells were transfected with pAc-per-V5-6XHis together with an empty plasmid (pMT-FH; FH denotes 3XFLAG-6XHis), pMT-ck1α-FH, or pMT-ck1α(K49R)-FH. Protein extracts were either analyzed directly by Western blotting (A, left), with detection of HSP70 as loading control, or subjected to lambda phosphatase treatment before Western blotting analysis (A, right), or visualized using Phos-Tag SDS-PAGE (B). C, Quantification of phosphorylated PER as shown in B using ImageLab (Bio-Rad). Following quantification, the amount of phosphorylated PER for each condition was presented as a fraction of the total PER isoforms present. Error bars indicate SEM from three biological replicates. *p < 0.05. D, S2 cells were either co-transfected with pAc-per-V5-6XHis and pMT-ck1α-6Xcmyc or singly transfected with pAc-per-V5-6XHis or pMT-ck1α-6Xcmyc. Protein extracts were divided into equal aliquots and each aliquot was independently incubated with α-cmyc beads, α-HA beads, or α-V5 beads (Sigma-Aldrich). Immunocomplexes were analyzed by Western blotting in the presence of the indicated antibody. The inputs (Ly) were also assayed. Arrow on the right panel indicates CK1α. Empty, unloaded lanes are present between lanes 4 and 5 and between lanes 10 and 11. Vu H. Lam et al. J. Neurosci. 2018;38:10631-10643 ©2018 by Society for Neuroscience