Mouse anti cul3

For immunoblotting of heads extract, 40 heads of male flies were collected and frozen at indicated time. Heads were homogenized with T300 buffer, and protein samples were loaded onto acrylamide gels. Proteins were transferred on nitrocellulose membranes (GE health), and the membranes incubated with rat anti-TANGO10 (Fig. 1 and SI Appendix, Fig. S4), mouse anti-TANGO10 (SI Appendix, Fig. S5), mouse anti-synapsin (Developmental Studies Hybridoma Bank 3C11), rat anti-GE-1 (73 (link)), mouse anti-CUL3 (BD Biosciences 611848), mouse anti-V5 (ThermoFisher), rat anti-HA (Sigma Aldrich), rabbit anti-PER (74 (link)), guinea pig anti-TIM (10 (link)), mouse anti-actin (Developmental Studies Hybridoma Bank, JLA20), and anti-FLAG (Sigma-Aldrich) antibodies. The blots were detected using enhanced chemiluminescence prime (GE Healthcare). Quantifications were performed using Image J from two separate experiments (SI Appendix, Fig. S5).