(A) Representative tracks of DD1320AA AnkB-mCherry vesicles in Ank2-/- MEFs (left). Scale bar, 10 µm. Tracks were plotted in an XY coordinate system assuming (0,0) as initial position (right). (B and D) Representative images of live Ank2-/- MEFs showing the localization of WT AnkB-mCherry to different organelles (B) and Pearson's co-localization coefficient (D). Organelle markers used include Rab5-GFP (early endosomes), LAMP1-GFP (lysosomes), Rab11-GFP (recycling endosomes), Mito-GFP (mitochondria), TGN38-GFP (Golgi). Scale bar, 10 µm. (C and E) Representative images of fixed WT MEFs showing the localization of the PI3P biosensor GFP-2×FYVE to different organelles (C) and Pearson's co-localization coefficient (E). Organelle markers detected using antibodies against endogenous proteins included EEA1(early endosomes), LAMP1 (lysosomes), Rab11 (recycling endosomes), Mito-Track (mitochondria), golgin-97 (Golgi). Data represent mean ± SD for three independent experiments. N = 16. Scale bar, 10 µm.