FIGURE 1.

PER is the stoichiometrically limiting factor in the circadian negative feedback loop in MEFs. A, robust bioluminescence rhythms in Per2^Luc MEFs. The bioluminescence rhythms were measured after a 2-h serum shock. B, circadian rhythms of endogenous clock proteins in MEFs. Per2^Luc MEFs were harvested at the indicated times after serum shock and immunoblotted. Note that there are four CLOCK isoforms: two hyperphosphorylated isoforms and two non- or hypophosphorylated isoforms (indicated by two asterisks), as has been shown in liver (14). Relative abundance of PER1 and PER2 was calculated from three different experiments (bottom panel). C, PER is the limiting factor among the clock proteins. Endogenous clock proteins in MEFs harvested at 16 and 24 h after serum shock were compared with known amounts of in vitro translated proteins on the same blot. Based on data in B and D, combined levels of PER1 and PER2 peak at 24 h after serum shock. 1× in vitro translated proteins is roughly equal to 1 fmol. D, relative abundance of endogenous clock proteins at 24 h in MEFs. Results are shown as means ± S.E. of three experiments. CLK, CLOCK; BM, BMAL1.