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. Author manuscript; available in PMC: 2010 Feb 10.
Published in final edited form as: Science. 2009 Oct 16;326(5951):437–440. doi: 10.1126/science.1172156

Fig. 1.

Fig. 1

Phosphorylation of S71 or S280 by AMPK destabilizes CRY1. (A) Human embryonic kidney (HEK) 293 cells expressing WT or mutant FLAG-CRY1 were treated with cycloheximide (CHX) as indicated. AA denotes CRY1 with both S71A and S280A mutations. (B) FBXL3-v5 and PER2 bound to FLAG-CRY1 were detected by immunoblot (IB) following immunoprecipitation (IP) from HEK 293 cells. (C) (Top) Alignment of the regions surrounding mouse (m) CRY1-S71 and ACC1-S79. (Bottom) In vitro kinase assays using FLAG-CRY1 purified from HEK 293 cells and purified AMPK (6). Phosphorylated CRY1 was detected by radiography (left) or IB using an antibody against phosphoACC1-S79 (right). (D) Phosphorylation of stably expressed FLAG-CRY1 by endogenous AMPK was detected by IB following IP from MG132-treated MEFs. (E) MEFs were treated with vehicle (−) or 2 mMAICAR (+) for 2 hours. (F) MEFs were treated with CHX ± 2 mM AICAR as indicated. CRY1 was detected by IP and IB for the FLAG epitope in (C to F). Graphs in (F) represent the means ± SD of three quantified IB samples per condition.