Clock gene expression rhythms during and after entrainment to daily temperature gradients. Canton-S flies were synchronized to a temperature gradient (16°C to 28°C) in constant darkness and then, during the experimental day, either kept in the temperature gradient or released to constant conditions at 25°C or 17°C. Fly heads were harvested 1, 5, 9, 13, 17, and 21 h after (subjective) peak temperature. Northern blot analyses for the clock-controlled transcripts tim, per, and vri (normalized to rp49 as an internal control) are represented in panel (A). For comparison the entrainment data set was plotted along with each of the two free run data sets in matching phase order. Each data point was the average of one to three independent experiments. Note that the entrained molecular phases were preserved during free run at either temperature, but that tim transcript levels appear to be down-regulated at constant 17°C. Western blot analysis for TIM, PER and ACTIN (as an internal control) of protein extracts from heads collected in the same schedule during entrainment and free run at constant 17°C (panel (B)) indicated that PER and TIM protein phases maintained a normal delay (~6 h) relative to transcript levels both during entrainment to the daily temperature gradient and subsequent free run at constant 17°C. Data for a representative Western blot are shown (note that, due to a lower amount of sample, the signals for the first time point, are artificially low).