FIGURE 9:

WHAMM binds to PI(3)P in vitro. (A) Different quantities of phospholipids immobilized on a nitrocellulose membrane were probed with purified MBP-WMD. Bound protein was detected using antibodies to MBP and visualized by chemiluminescence. MBP-WMD binding was quantified by measuring the mean pixel intensity of protein-specific spots after normalizing to background staining. (B) Different concentrations of PI(3)P immobilized on a nitrocellulose membrane were probed with purified GST-Rab1A. Bound protein was detected using antibodies to GST and quantified as in A. (C) Synthetic liposomes containing 5% of the indicated phosphoinositides were incubated with MBP and MBP-WMD and collected by centrifugation. Liposome-associated proteins were subjected to SDS–PAGE and detected by immunoblotting for MBP. (D) The amount of MBP-WMD pulled down was determined by densitometry. Each bar represents the mean (+ SD) from three to four experiments. (E) PI(3)P-containing biotinylated liposomes were mixed with Alexa Fluor 555–labeled MBP-WHAMM and Alexa Fluor 488–labeled streptavidin and examined microscopically.