Chemiluminescent western blotting has been in common practice for over three decades, but its use as a quantitative method for measuring the relative expression of the target proteins is still debatable. This is mainly due to the various steps, techniques, reagents, and detection methods that are used to obtain the associated data. In order to have confidence in densitometric data from western blots, researchers should be able to demonstrate statistically significant fold differences in protein expression. This entails a necessary evolution of the procedures, controls, and the analysis methods. We describe a methodology to obtain reliable quantitative data from chemiluminescent western blots using standardization procedures coupled with the updated reagents and detection methods.

We have identified a novel component of the circadian clock that regulates its sensitivity to light at the evening light to dark transition. USP2 (Ubiquitin Specific Protease 2), which de-ubiquitinylates and stabilizes target proteins, is rhythmically expressed in multiple tissues including the SCN. We have developed a knockout model of USP2 and found that exposure to low irradiance light at ZT12 increases phase delays of USP2−/− mice compared to wildtype. We additionally show that USP2b is in a complex with several clock components and regulates the stability and turnover of BMAL1, which in turn alters the expression of several CLOCK/BMAL1 controlled genes. Rhythmic expression of USP2 in the SCN and other tissues offers a new level of control of the clock machinery through de-ubiqutinylation and suggests a role for USP2 during circadian adaptation to environmental day length changes.

Chick pineal cells in culture display a circadian rhythm of melatonin release. Light pulses can entrain (phase shift) the rhythm. One candidate for the photoentrainment pathway uses a mitogen-activated protein kinase (MAPK), also known as extracellular signal-regulated kinase (ERK). We tested the hypothesis that activation of ERK (by phosphorylation to p-ERK) is necessary and/or sufficient for entrainment by measuring the ability of several drugs, light, and other perturbations to change levels of p-ERK and to induce phase shifts in the melatonin rhythm. If changes in the levels of p-ERK are sufficient for photoentrainment, then all perturbations that reduce its level must induce light-like phase shifts, and all those that increase its level must induce dark-like phase shifts. If such changes are necessary for photoentrainment, then light pulses must reduce p-ERK levels, and the duration of the light pulse, the magnitude and duration of the change in p-ERK, and the size of the phase shift must correlate. We found five perturbations that reduced p-ERK levels. Of these, two induced light-like phase shifts (PD 98059 and caffeine), one induced dark-like phase shifts (SB203580), and two did not induce phase shifts at all (U0126 and omitting a medium change). Serum increased p-ERK levels without inducing any phase shifts. Finally, light pulses did not elicit changes in p-ERK, nor was there a diurnal rhythm in p-ERK levels, nor could rapid changes in p-ERK levels have accounted for duration effects of light pulses on phase shifts. Taken together, these results argue strongly against the hypothesis that reduction (or increases) in MAPK/ERK activation is necessary or sufficient for entrainment in chick pineal cells.