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. 2014 Feb 17;124(3):1057–1068. doi: 10.1172/JCI71863

Figure 4. LC-CoAs compete with phosphoinositides for NHE1 binding in vitro.

Figure 4

NHE1 cytosolic domain (cNHE1) binding to LC-CoAs and phospholipids was determined by membrane overlay assays, as described in Methods. (A) Map of LC-CoAs and phosphoinositides spotted on nitrocellulose membranes. (B) cNHE1 incubation with membranes spotted with LC-CoAs and phosphoinositides, followed by anti-NHE1 antibodies, and then HRP-conjugated IgG, as previously described (10). Membrane shown is representative of n = 4. (C) Map of LC-CoAs and phosphoinositides spotted on nitrocellulose membranes for competition assays. (D) Competition with PI(4,5)P2 for NHE1 cytosolic domain binding to LC-CoAs. cNHE1 incubated overnight at 4°C. Binding buffer only for additional 12 hours (left panel); 25 μM PI(4,5)P2 in binding buffer over the second 12-hour period (right panel). Detection of cNHE1 binding as in B. Membrane shown is representative of n = 3. (E) Map of spotted membrane phospholipids. LPA, lysophosphatidic acid; LPC, lysophosphatidylcholine; PI, phosphatidylinositol; PE, phosphatidylethanolamine; PC, phosphatidylcholine; S-1P, sphingosine-1-phosphate; PA, phosphatidic acid; PS, phosphatidylserine. (F) Competition with palmitoyl-CoA for NHE1 cytosolic domain binding to phosphoinositides. cNHE1 incubated overnight at 4°C. Binding buffer only for additional 12 hours (left panel); 25 μM palmitoyl-CoA in binding buffer over the second 12-hour period (right panel). Detection of cNHE1 binding to phospholipids, as in B and D. Data are representative of n = 5.