Decreased GSK3β activity through downregulation of P-Tyr216 is related to increased axonal growth. (A) Representative images of dorsal column fibers traced with cholera toxin B in sagittal sections following SCI (dorsal hemisection) or conditioning lesion (CL) in rats; r: rostral; c: caudal; d: dorsal; v: ventral. Dashed lines label the border of the glial scar. Scale bar: 100 μm. (B) Representative images of neurite outgrowth of naïve and conditioned rat DRG neurons (CL). Scale bar: 50 μm. (C) Quantification of B; (n = 40 to 92). (D) Western blot analysis of P-AktSer473, P-GSK3βSer9 and P-GSK3βTyr216 on DRG extracts from uninjured rats (Un) or subjected to SCI or CL. (E) Quantification of D; (n = 3 to 4). (F) Western blot analysis of P-GSK3βSer9 and P-GSK3βTyr216 in SCI site extracts from rats with SCI or conditioning lesion (CL); (G) Quantification of F; (n = 5 to 7). (H) P-GSK3βTyr216 and GSK3β immunostaining of the growth cone of naïve rat DRG neurons or conditioned (CL) neurons; (n = 10 to 12). Scale bar: 4 μm. (I) Western blot analysis of P-CRMP-2 on SCI site extracts from rats with SCI or conditioning lesion (CL); (n = 4 to 7). (J) Western blot analysis of P-FynTyr530 on SCI site extracts from rats with SCI or conditioning lesion (CL); (n = 4 to 5). (K) P-GSK3βTyr216 and GSK3β staining of the growth cone of control transfected DRG neurons or neurons transfected with a Fyn kinase dead construct (Fyn KD); (n = 72 to 101). Scale bar: 3 μm. All error bars are SEM. *P <0.05. **P <0.01. ***P <0.001. Two-tailed Student’s t test. CRMP-2, collapsin response mediator protein 2; DRG, dorsal root ganglia; GSK3β, glycogen synthase kinase 3β; SCI, spinal cord injury; SEM, standard error of the mean.