CRY Drives Cyclic CK2-Mediated BMAL1 Phosphorylation to Control the Mammalian Circadian Clock
Fig 5
Identification of CRY1 and BMAL1 regions involved in CRY-enhanced BMAL1–CK2β binding.
(A) Schematic representation of Split Luc mBMAL1 deletion mutant expression vectors. (B) Cos7 cells were transfected with ELucN-CK2β and either full length (WT) or mutant (Bd1-5) ELucC-BMAL1, in combination with either pcDNA (-; negative control), Myc-HA-CRY1, or Myc-CRY2. Expression of ELucC-BMAL1-full length and deletion mutants (Bd1-5) was confirmed by immunoblot analysis using anti-His antibody (lower panel). BMAL1–CK2β binding was monitored by a real-time bioluminescence imaging (upper panel). Shown are averaged peak bioluminescence values (n = 6 experiments). Error bars indicate SD. (C) Schematic representation of mCRY1 deletion mutant expression vectors. (D) Cos7 cells were transfected with ELucN-CK2β and ELucC-BMAL1, in combination with either full length (WT) or mutant (Cd1-4) pcDNA-FLAG-CRY1. Expression of CRY1-full length and deletion mutants (Cd1-4) was confirmed by immunoblot analysis using FLAG antibody (lower panel). BMAL1–CK2β binding was monitored by a real-time bioluminescence imaging (upper panel). Shown are averaged peak bioluminescence values (n = 6 experiments). Error bars indicate SD.