FIG. 4.

(A) F10 is tightly associated with membranes during wt infections. Cells were left uninfected (u) or infected with wt virus at an MOI of 5 and were harvested at 8 hpi. A PNS was prepared and subjected to either TX-114 or sodium carbonate (Na₂CO₃) extraction as described in Materials and Methods. Equivalent amounts of the aqueous and detergent phases (TX-114 extraction) and the supernatant and pellet fractions (Na₂CO₃ extraction) were resolved by SDS-12% PAGE and transferred to a nitrocellulose filter. Immunoblot analysis was performed with the anti-F10 antiserum. The lower half of the blot was probed with an antiserum specific for a known viral membrane protein (D8) as a control for extraction conditions. The F10 protein partitioned into the detergent phase upon TX-114 extraction of the PNS and into the pellet after Na₂CO₃ extraction of the PNS. (B) F10 binds phosphoinositides in vitro. PIP strip membranes containing immobilized phosphoinositides (Echelon Biosciences) were incubated overnight with 80 ng of purified FLAG-F10 protein/ml (middle and right blots) or with the control protein 3XFLAG-BAP (left blot). For visualization of the bound FLAG-F10, an immunoblot analysis was then performed with the anti-FLAG antibody (left and right blots), an appropriate secondary antibody, and chemiluminescent development. The immobilized species were as follows: LPA, lysophosphatidic acid; LPC, lysophosphocholine; PI, phosphoinositide; PI(3)P, phosphoinositide-3-phosphate; PI(4)P; PI(5)P; PE, phosphatidylethanolamine; PC, phosphatidylcholine; S1P, sphingosine-1-phosphate; PI(3,4)P₂; PI(3,4)P₂; PI(4,5)P₂; PI(3,4,5)P₃; PA, phosphatidic acid; and PS, phosphatidylserine. The chemiluminescent signals were quantitated, and the hierarchy of binding is shown schematically as follows: highest binding, spots 1, 2, 3, and 5 (white text in black boxes); strong binding, spots 4 and 6 (gray boxes); modest binding, spot 7 (white box); and minimal binding, spot 8 (dashed box).