Interaction of ArfGAP-PH with full length GxcDD and phosphoinositides. (A) GST-ArfGAP-PH was expressed in E. coli and bound to glutathione-sepharose beads. Beads were incubated with wild type cell lysates (P). GST bound beads were used as a control (C). Pulldown eluates were resolved by 10% SDS-PAGE, western blots revealed GxcDD as an interacting protein. (B) GST-ArfGAP-PH was cleaved by thrombin to liberate the ArfGAP-PH protein and purified protein was tested for oligomerization using increasing amounts of glutaraldehyde as a crosslinking agent (0 – 0.1% v/v). (C) The CH domain has the potential to bind to the GEF domain and the ArfGAP-PH domain. Lysates from cells expressing GFP-CH domain (GFP-CHD) were used in pull down assays employing GST-RacGEF or GST-ArfGAP-PH bound to glutathione-sepharose beads. The blot was probed with a GFP-specific monoclonal antibody. (D) PtdIns(3,5)P2, PtdIns(4,5)P2, PtdIns(3,4,5)P3 were spotted on a PVDF membrane and incubated with ArfGAP-PH protein and binding detected using GxcDD specific polyclonal antibodies.
Mondal et al. BMC Cell Biology 2007 8:23 doi:10.1186/1471-2121-8-23