α clk

Flies were entrained for three full days in 12 hr light:12 hr dark (LD) conditions at 25°C and collected on the fourth day at the indicated time points (ZT) and frozen immediately on dry ice. Heads were separated using frozen metal sieves and homogenized in 3x volume of RBS buffer (20 mM HEPES at pH 7.5, 50 mM KCl, 10% glycerol, 2 mM EDTA, 1 mM DTT, 1% Triton X-100, 0.4% NP-40, 10 μg/mL aprotinin, 5 μg/mL leupeptin, 1 μg/mL pepstatin A, 0.5 mM PMSF, 1X PhoStop (Roche)) [46 (link), 51 (link)]. Homogenate was sonicated using a Fisher Scientific sonicator for five seconds and repeated five times with 10-second pauses in between. Samples were spun down at 14,000 rpm for 15 minutes at 4°C to remove cell debris. Supernatant was collected, transferred to new tubes, and spun down again for 10 minutes at 14,000 rpm at 4°C. Supernatant was collected and protein levels were quantified using a spectrophotometer (Eppendorf). Proteins were resolved by SDS-PAGE (Criterion 5% gels, Bio-Rad), transferred to nitrocellulose membranes (Bio-Rad) and incubated in 5% blocking solution (Bio-Rad) in 1XTBST with α-PER (GP5620) (1:2000), α-TIM R3 (1:2000), or α-CLK (Santa Cruz) (1:1000). Membranes were imaged and protein levels were quantified using the ChemiDoc MP system with Image Lab software (Bio-Rad).

Transgenic flies expressing BRM fused to FLAG epitope tags were entrained in 12 hr light:12 hr dark (LD) conditions at 25°C for three days and collected on the fourth day. Fly head collection and protein extraction with RBS buffer with sonication were performed as described above. Extracts were quantified and equal concentrations were subjected for IP. Samples were pre-cleared using sepharose beads (Sigma) to reduce nonspecific binding. Co-IPs were performed as described for S2 cell experiments except α-FLAG M2 (Sigma), α-PER (GP5620), α-TIM (R3), and α-CLK (Santa Cruz Biotechnology H3107) antibodies were used. Samples were incubated with antibodies for 4 to 6 hours at 4°C on an end-over-end rotator. 20 μl of GammaBind Plus sepharose beads (GE) was added and incubation was continued for 2 hours. Samples were washed with RBS buffer three times, 10 minutes each, and immune complexes were resolved by SDS-PAGE as described above.