Fig. 2. Analysis of phosphoinositide binding specificities of GFP-FENS-1 (A) and Myc-DFCP1 (B,C) constructs expressed in COS-7 cell lysates using lipid competition for binding to phosphoinositide-derivatised beads. The indicated GFP-tagged FENS-1 constructs or Myc-tagged DFCP1 constructs were expressed in COS-7 cells and lysates prepared as described in the Materials and Methods. Aliquots of these lysates were mixed with the indicated phosphoinositides (5 µM and 10 µM for FENS-1 and DFCP1, respectively) in a total volume of 1 ml for 10 minutes on ice, then transferred onto 15 µl packed PtdIns(3,4)P2-derivatised beads and mixed end to end at 4°C for 45 minutes. Beads were washed briefly four times and the recovery of the relevant proteins was assessed by SDS PAGE and the appropriate Western-blotting procedure (see Materials and Methods). 1% aliquots of the various lysates were taken before mixing with the beads to assess relative expression levels and recovery on the beads of each construct.
