DNA constructs and S2 culture

Constructs used were as follows; pAc5.1-Flagx3, pAc5.1-Myc-Pk, pAc5.1-Dsh-GFP, pAc5.1-Scrib-PDZ-3-4-HA and pAc5.1-HA-Dgo (all gifts from Dr. Jenny, AECOM, USA) and pAc5.1-Vang-Flagx3 [44]. GFP-TZ was used as a control, and is the Drosophila ciliary protein Mks1. pAc5.1-Vang-Flagx3-Y374A, FKYY371AAYA, Y374F and V376A were generated though site-directed mutagenesis. C-terminal truncations of Vang were generated through PCR and cloned into pAc5.1-Flagx3 using NotI-XbaI restriction sites. All primers used are available upon request.

Unless otherwise stated, lysates were prepared from S2 cells. S2 cells were maintained according to standard protocols, and were grown in Schneider’s Medium (Gibco) supplemented with 10% heat-inactivated Fetal Bovine Serum (Gibco). Cells were plated in 12 well plates at a dilution of 1.5x10⁶ and were transfected with the indicated constructs using Effectene (Qiagen) according to manufacturer’s protocols. Cells were lysed ~48 hrs later in buffer containing 50mM Tris-HCl pH 7.5, 150mM NaCl, 1mM EDTA and 1% Triton-X-100.