To perform co-affinity purification experiments, cloned ORFs were transferred from pDONR207 to pDEST27 expression vector (Invitrogen) to achieve GST fusion, and to pCI-neo-36FLAG vector [61] for 36FLAG-fusion. Cell transfections were performed using Lipofectamine 2000 (Invitrogen). Unless specified otherwise, 5610 5 HEK-293T cells were dispensed in each well of a 6-well plate, and transfected 24 h later with 600 ng of each plasmid DNA per well. Two days post transfection, HEK-293T cells were washed in PBS, then resuspended in lysis buffer (0.5% Nonidet P-40, 20 mM Tris-HCl at pH 8, 120 mM NaCl and 1 mM EDTA) supplemented with Complete Protease Inhibitor Cocktail (Roche). Cell lysates were incubated on ice for 20 min, and then clarified by centrifugation at 14,0006g for 10 min. For pull-down analysis, 400 mg of protein extracts were incubated for 1 h at 4uC with 25 ml of glutathione-sepharose beads (Amersham Biosciences) to purify GST-tagged proteins. Beads were then washed 3 times in ice-cold lysis buffer and proteins were recovered by boiling in denaturing loading buffer (Invitrogen).