Cell and baculovirus culture

S2 and Sf9 cell lines (kind gift from Peter Becker, Munich) were maintained at 26 °C in Schneider medium (Gibco) and Sf-900 medium (Gibco), respectively, supplemented with 10% fetal calf serum. RNA interference, baculovirus generation and infection are described in ref. ⁴⁰. Briefly, double-stranded RNA was generated by T7 Polymerase in vitro transcription from PCR amplimers generated with T7 promotor-containing primers (Supplementary Table 1). Double-stranded RNAs were transfected into S2 cells using Effectene (Qiagen). Baculoviruses were generated using the Bac-to-bac system (Invitrogen). Baculoviruses were amplified twice and then used to infect Sf9 cells for protein production. Cells were then harvested 48–72 h after infection. EcR (ER33854) and USP (LD09973) cDNAs were obtained from BDGP. Vectors for generation of baculoviruses expressing untagged EcR, N-terminally FLAG-tagged EcR and N-terminally HA-tagged USP were generated by PCR-cloning of the respective open-reading frames into pFastBac or pVL1392 using appropriate sets of primers. Baculoviruses and expression vectors for dLint-1 and dMi-2 were constructed in the same manner^46,47.