Figure 7

Increased microtubule growth in GSK3β depleted neurons is related to decreased CRMP-2 phosphorylation. (A) MAP1B, CLASP2 and CRMP-2 western in CAD cells. (B) Microtubule growth in rat neurons transfected with EB3-mCherry alone (control) or co-transfected with WT MAP1B, ST/AA-MAP1B or ST/DD-MAP1B. (C) Intensity ratios between acetylated and tyrosinated microtubules in growth cones from rat DRG neurons transfected with EB3-mCherry alone (control) or co-transfected with WT MAP1B, ST/AA-MAP1B or ST/DD-MAP1B. (D) Representative images of C. Scale bar: 3 μm. (E) Intensity ratios between acetylated and tyrosinated microtubules in growth cones from rat naïve DRG neurons either non-infected (control) or infected with WT CLASP2, 9S/A-CLASP2 or 8S/D-CLASP2. (F) Representative images of E. Scale bar: 3 μm. (G) Microtubule growth in neurons transfected with EB3-GFP alone (control) or co-transfected with WT CRMP-2, T/A-CRMP-2, or T/D-CRMP-2. (H) Intensity ratios between acetylated and tyrosinated microtubules in growth cones from neurons transfected with EB3-GFP alone (control) or co-transfected with WT CRMP-2, T/A-CRMP-2, or T/D-CRMP-2. (I) Representative images of H. Scale bar: 5 μm. (J) Microtubule growth in naïve neurons from cre⁺GSK3βwt/wt mice or cre+GSK3βlox/lox mice transfected with EB3-mCherry, or cre⁺GSK3βlox/lox mice co-transfected with EB3-mCherry and T/D-CRMP-2 (cre⁺GSK3βlox/lox + T/D-CRMP-2). (K) Intensity ratios between acetylated and tyrosinated microtubules in growth cones from cre⁺GSK3βwt/wt, cre+GSK3βlox/lox or cre⁺GSK3βlox/lox transfected with T/D-CRMP-2 (cre⁺GSK3βlox/lox + T/D-CRMP-2). (L) Representative images of K. Scale bar: 5 μm. All error bars are SEM. *P <0.05. **P <0.01. ***P <0.001. ****P <0.0001. Two-tailed Student’s t test. CLASP2, cytoplasmic linker associated protein 2; CRMP, collapsin response mediator protein; DRG, dorsal root ganglia; GSK3β, glycogen synthase kinase 3β; MAP1B, microtubule-associated protein 1B; SEM, standard error of the mean. WT, wild type.