AnkB’s ZU5^C domain interacts with membrane PtdIns(3)P. (A) Coomassie blue staining and immunoblots of purified AnkB proteins. (B) Image shows the known crystal structure of the ZU5^N-ZU5^C-UPA-DD supermodule of AnkB and highlights a positively charged surface (yellow) within the ZU5^C domain. High magnification view of the boxed region depicts all residues mutated for analysis, including those found to be essential for AnkB binding to PtdIns(3)P lipids (red dotted lines). (C) Protein–lipid overlay assay showing affinity of the ZU5-DD region for PtdIns(3)P. IB, immunoblot; LPA, lysophosphatidic acid; LPC, lysophosphatidylcholine; PE, phosphatidylethanolamine; PC, phosphatidylcholine; PA, phosphatidic acid; PS, phosphatidylserine; S1P, sphingosine-1-phosphate. (D) Binding of WT 220-kD AnkB, WT ZU5^N-ZU5^C-UPA-DD, and the ZU5^C mutant proteins to polyPIPosomes bearing the indicated PtdInsPs measured through ELISA. The data shown are for one experiment out of three repeats. Data represent mean ± SEM. RU, relative unit. (E) Time-lapse image and kymographs of AnkB-mCherry proteins and synaptophysin-YFP localization and dynamics in axons.