Phospho-CRMP2 (Thr514) Antibody - #AF3459

Fig. 6Sema3F/Npn-2 signaling regulates CRMP2 phosphorylation. A Reduction of CRMP2 phosphorylation in Npn-2 knockdown tissues. Adult rat hippocampi injected with various viruses as indicated were subjected to western blot with different phospho-CRMP2 antibodies. The levels of p-S522, p-T514, and p-T555-CRMP2 were significantly reduced in Npn-2 knockdown animals compared with the control group while the expression of CRMP2 was not changed, and the knockdown effects were rescued by the expression of hNpn-2. B–E Quantitation of A. n = 4, *P < 0.05, ***P < 0.001. F Semaphorin induced CRMP2 phosphorylation in primary cultures. Primary cultured neurons were treated with 5 nM AP, AP-Sema3F, or AP-Sema3A for 12 h. CRMP2 phosphorylation levels were detected by various phospho-CRMP2 antibodies as indicated using western blot. Sema3F treatment upregulated the levels of p-S522, p-T514, and p-T555-CRMP2 while made no effect on CRMP2 expression. G–J Quantitation of F. n = 4, *P < 0.05, **P < 0.01. One-way ANOVA, post hoc Tukey test. Error bars represent SEM. K Time course of Sema3F regulating CRMP2 phosphorylation. Primary cultured neurons were treated with 5 nM AP-Sema3F for 5 min, 30 min, 60 min, 6 h, 12 h, and 24 h. Levels of CRMP2 phosphorylation were detected by various phospho-CRMP2 antibodies as indicated using western blot. The levels of p-S522-CRMP2, p-T514-CRMP2, and p-T555-CRMP2 increased over time and peaked at around 12 h, while CRMP2 expression was not significantly changed. L Quantitation of K (n = 3). M Sema3F that regulates CRMP2 phosphorylation is dose-dependent. Primary cultured neurons were treated with AP-Sema3F at various doses as indicated for 12 h. CRMP2 phosphorylation levels were detected by phospho-CRMP2 antibodies as indicated using western blot. The levels of p-S522-CRMP2, p-T514-CRMP2, and p-T555-CRMP2 were increased over increased doses and peaked around 0.5 nM, while the expression of CRMP2 was not significantly changed. N Quantitation of M (n = 3)

Fig. 5. | The interaction between GSK3 and CRMP2 was validated. A) CRMP2 was detectable by immunoblotting coupled with co-IP.Representative immunoblots (B) and quantification expression of GSK3, CRMP2, and their phosphorylation levels in N2a-APP cells treated with aFGF for 24 h (C-F).