Mammalian cell overexpression and siRNA depletion

Yip3 was cloned into the mammalian vector pcDNA3.1. HEK293 cells were transfected with pcDNA3.1 human Yip3 or pcDNA3.1, collected 42 h later, lysed by osmotic shock and shearing, and membranes were purified by ultracentrifugation. Membrane recruitment was as published in buffer plus 100 mM (NH4)2SO4, 100 mM GTPgS, 0.1 mg ml 21 BSA, protease inhibitor cocktail (0.34 units ml aprotinin, 0.01 mg ml leupeptin, 1 mM pepstatin A), 0.2 mM DTT and ATP regeneration system. Rab9–GDI complex (300 ng, 4.1 pmol) and membranes were mixed in 250 ml for 40 min at 37 8C. Ice-cold buffer (64 mM HEPES-KOH, pH 8.0, 100 mM NaCl, 8 mM MgCl2, 2 mM EDTA; 0.5 ml) was added and each reaction was then layered over 0.4 M sucrose þ ice-cold buffer and spun for 10 min at 342,000g, 2 8C in a TLA-100.2 rotor (Beckman). Pellets were analysed by 12.5% SDS–PAGE and anti-Rab9 immunoblot. HeLa S-3 cells were transfected with 0.13 mM siRNA duplex (sense 5 0 -(GCUUGUG CUCUUUGGCCGA)d(TT)-3 0 ; Dharmacon) using Oligofectamine (Invitrogen). After 77 h, cells were lysed in 25 mM HEPES pH 7.3 with protease inhibitors by 10 passages through a 25G needle. Extracts were spun 30s at 1,000 g; supernatants were centrifuged 15 min, 321,000g in a TLA 120.1 rotor (Beckman). The membrane pellet was washed in PBS.