Figure 3.

PA Binds to PID.

(A) Immunoblot of His-tagged PID, PID2, WAG1, and WAG2 produced in E. coli Rosetta (DE3).

(B) Binding of PA to PID, PID2, WAG1, and WAG2. The D330 and D100 Arabidopsis RbohD fragments were used as positive and negative controls, respectively.

(C) and (D) ELISAs of the binding of PID, PID2, WAG1, and WAG2 to PA (C) and PC (D). Data are means ± sd of three replicates.

(E) and (F) Binding of PID to the indicated lipids (10 μg) on nitrocellulose filters. DAG, diacylglycerol; PS, phosphatidylserine; PI, phosphatidylinositol; PG, phosphatidylglycerol; PE, phosphatidylethanolamine; PC, phosphatidylcholine; PA, natural PA from soybean; PtdIns3P, phosphatidylinositol 3-phosphate; PtdIns4P, phosphatidylinositol 4-phosphate; PtdIns5P, phosphatidylinositol 5-phosphate; PtdIns(3,4)P₂, phosphatidylinositol-3,4-bisphosphate; PtdIns(3,5)P₂, phosphatidylinositol-3,5-bisphosphate; PtdIns(4,5)P₂, phosphatidylinositol-4,5-bisphosphate. PID binding to lipids was assayed by immunoblot analysis using an anti-His antibody. The protein from the pET28a vector was used as a control.

(G) Binding of PID to the following PA species: dipalmitoyl PA (di16:0), distearoyl PA (di18:0), dioleoyl PA (di18:1), dilinoleoyl PA (di18:2), palmitoyl-oleoyl PA (16:0-18:1), palmitoyl-linoleoyl PA (16:0-18:2), stearoyl-oleoyl PA (18:0-18:1), stearoyl-linoleoyl PA (18:0-18:2), and natural PA from soybean (Soy).

(H) Dose-dependent binding of PID to natural PA.