Fig. 2

pS25- and pS1201 MAP 1B bind to axonal MTs. a-h Immunostaining images of Tuj1 (blue) /phalloidin (red) /pS25 (green: b-d), or pS1201 (green: f-h) in the axonal growth cone of 3DIV mouse cortical neurons. High magnificent images in ROI (a or e) are shown (b-d) or (f-h), respectively. Images of normalized mean deviation product (nMDP) (d, (h; see Materials and Methods) from the double staining, Phalloidin/pS25 (c) and Tuj1/pS25 (d); phalloidin/pS1201 (g) and Tuj1/pS1201 (h). Tuj1/pS25 and Tuj1/pS1201 show strong co-localization in axons (cyan in the merged images and hot colors in the nMDP images), although both phalloidin/pS25 and phalloidin/pS1201 show weak or no colocalization (cold colors in the nMDP images). Scale bars, 3 μm. i Quantification of colocalization between the phosphorylated MAP 1B (pS25 or pS1201) and cytoskeletons (tubulin and F-actin) in the axonal growth cone (n = 10 for each; ***p < 0.001). Data are presented as mean ± SD. Student’s t-test. j Determination of MT binding affinities by the co-sedimentation assay, using an extract of E15 mouse brains. Ex: extract; P: pellets; and S: supernatant