Fig 1. Binding of recombinant EhLTP (rEhLTP) to lipids by protein lipid overlay assay.

(A) Membrane Lipid Strips™ were incubated overnight at 4°C with 1 mg/ml of rEhLTP1. (B) Membrane Lipid Strips™ were incubated overnight at 4°C with 1 mg/ml of rEhLTP3. Binding of protein to the lipid spots was detected by subsequent incubation with anti-His mouse monoclonal antibody and secondary anti-mouse peroxidase antibody as described in Methods. The lipids spotted on the strips are: Glyceryl tripalmitate or Triglyceride (GT), Diacylglycerol (DAG), Phosphatidic Acid (PA), Phosphatidylserine (PS), Phosphatidylethanolamine (PE), Phosphatidylcholine (PC), Phosphatidylglycerol (PG), Cardiolipin (CL), Phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PtdIns4P), phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P₂], phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P₃)], Cholesterol, Sphingomyelin (SM), 3-sulfogalactosylceramide or Sulfatide (SGC), and Blank. C Negative control, the same treatment as “A” or “B”, the only exception was in place of study proteins (rEhLTP3 or rEhLTP1), we used rEhNAD kinase, which has no reported lipid binding ability.