Figure 3

Pull-down assays by using oligonucleotides. (A) Both BMAL1 and CLOCK proteins bind to Per2 oligonucleotide. BMAL1 is phospholrylated in a circadian manner, whereas no binding to the random oligonucleotide occurs. Mouse liver lysates at CT 16 were electrophoresed and immunoblotted with anti-BMAL1 and anti-CLOCK (PC) and immunoprecipitated by anti-CLOCK and blotted by either BMAL1 or CLOCK (IP). WT; wild-type hPer2 oligonucleotide, E1; E1 mutant oligonucleotide, E2; E2 mutant oligonucleotide, E1mE2m; E1, E2 double mutant oligonucleotide. (B) As for BMAL1, the binding to E2m is clearly observed; whereas no binding to either E1m or E1mE2m is detected. For CLOCK, the binding to E2m is observed; however, the binding to E1m is hardly seen and no binding to E1mE2m is evident.