CULLIN-3 Controls TIMELESS Oscillations in the Drosophila Circadian Clock
Figure 7
TIM interactions with CUL-3 and SLMB.
(A) Anti-CUL-3-FLAG immunoprecipitation from head extracts followed by anti-TIM immunoblotting in per+ (left) and per0 (right) backgrounds. Slots 1 and 3 are negative controls (faint band reveals low non-specific anti-FLAG background), and 50 µg of total proteins were used for per+ and per0 input extracts. Flies were entrained 3 d in LD and transferred to DD for collection at CT15. (B) Left: anti-FLAG-SLMB immunoprecipitation from head extracts collected at different time points, followed by anti-TIM immunoblotting. Two consecutive time points were pooled for IPs in a per+ background. Slot 7 is the negative control, and 50 µg of extracts were used for per+ input. Right: anti-TIM Western blot of head extracts of w; tim-gal4 (50 µg) and per0 w; tim-gal4 (25 µg) indicate that TIM levels are much higher in a per0 background. At least two independent experiments were done for each assay with very similar results.