Fig 2. Lipid binding activity of Ysc84-Nt.

(A) Dot-blot assays were performed on PIP strips to determine the specificity of Ysc84 wild-type and mutants against 15 phospholipids. The membrane was incubated with 10 μM of His-tagged purified protein and proteins detected using anti-His tag antibodies. (B) Liposome co-sedimentation assay was performed using Ysc84-Nt and 70% PE, 30% PC-based liposomes supplemented in right lanes with 10% of PI(4,5)P₂. Proteins in the supernatant (S) and pellet (P) were visualized by Coomassie staining. Densitometry was used to determine the proportion of protein pelleting with liposomes (lower panel). Results are mean (±SD) of two independent experiments. Kinetics of F-actin barbed end elongation in the absence and presence of Ysc84 and PI(4,5)P₂. (C,D) In a pyrene-based fluorimetry assay pre-formed actin seeds (1 μM) were mixed with 1 μM G-actin and incubated with wild type and KK (C) or wild type and LK (D) Ysc84-Nt in the absence and presence of 1.2 μM PI(4,5)P₂.