Normal axonal cytoskeleton structure in AnkB-deficient neurons. (A and B, top) 3D-STORM images of β2-spectrin structure in DIV10 AnkB^+/+ (A) and AnkB^−/− (B) axons. Violet to red color-coded z position is shown, representing closest to and farthest from the substratum, respectively. (middle) Graphs show the distribution of molecules along the axon long axis. (bottom) Fourier transform of the 1D localization distributions shows a fundamental frequency of 180 nm⁻¹ and an overtone. Data are representative of axonal STORM images and analysis from one experiment out of three repeats. For the experiment shown, n = 10. (C) Expression and localization of total and 440-kD AnkB, β2-spectrin, and ankyrin-G proteins (AnkG) and MAP2 in DIV8 AnkB^+/+ and AnkB^−/− hippocampal neurons. Arrowheads indicate axon initial segments. Color arrows represent the direction of coordinate planes in the rotated 3D image. (D) Immunoblots show ankyrin-G (all neuronal isoforms), β2-spectrin, β4-spectrin, and actin expression in PND2 brains. All experiments were repeated three times.