Analysis of the interaction between cytohesin 2 and IPCEF1.

IPCEF1 has been reported to interact with ADP-ribosylation factor GTP exchange factors of the cytohesin family and function by modulating the cytohesin 2 activity. This article describes methods used to study the interaction and activation of cytohesin GEFs by IPCEF1. The experimental approaches described here include physical and functional interaction assays by which the association of IPCEF1 with cytohesin 2 is explored both in vitro and in vivo. The methods used to analyze the physical association include GST-pull down and coimmunoprecipitation approaches. We also used yeast two-hybrid and colocalization assays to study the interaction between IPCEF1 and cytohesins. The functional relationship between IPCEF1 and cytohesin 2 was assessed by studying the effect of IPCEF1 on the in vitro and in vivo stimulation of ADP-ribosylation factor 6 GTP formation by cytohesin 2.