Figure 4
Protein-lipid overlay assay of profilins. Phosphoinositide (PIP) overlay assay of Glutathione-S-transferase (GST)-fusion proteins showed selective binding of GST-PFN1, GST-PFN2, and GST-PFN4 to nitrocellulose-bound phosphoinositides (100 picomoles per spot). GST alone did not bind. PIP strips™ were incubated with 0.5 μg/ml of each fusion protein as indicated. Layout of strips (according to Molecular Probes product information) was as follows. Spot # 1: Lysophosphatidic acid; # 2: Lysophosphatidylcholine; # 3: Phosphatidylinositol (PtdIns); # 4: PtdIns(3)P; # 5: PtdIns(4)P; # 6: PtdIns(5)P; # 7: Phosphatidylethanolamine; # 8: Phosphatidylcholine; # 9: Sphingosine 1-phosphate; # 10: PtdIns(3,4)P2; # 11: PtdIns(3,5)P2; # 12: PtdIns(4,5)P2; #13: PtdIns(3,4,5)P3; #14: Phosphatidic acid; #15: Phosphatidylserine; #16: Blank.