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. 2018 Jul 25;8:135. doi: 10.1038/s41398-018-0182-y

Fig. 5. Rescue of altered EB3 expression and tubulin polymerization induced by brain-specific Ank3 repression is blocked by inhibition of CRMP2.

Fig. 5

a Schematic representation of the experimental design. Mouse neuro-2a cells were transfected with pHAGE-EF1α-dCas9-KRAB repressor plasmid and sgRNA(MS2)_EF1α plasmid expressing either the non-targeting control sgRNA or the sgRNA targeting Ank3 exon 1b, followed by puromycin and zeocin selection. Cells were subsequently treated with 5 µM lacosamide (LCM) for 24 h, followed by treatment with lithium (1 mM) or CHIR99021 (1 µM) for 1 h, and cell harvest for protein extraction. b Top: Representative western blot of EB3 and GAPDH. Bottom: Quantification of EB3 expression normalized to GAPDH. Univariate ANOVA, LCM effect F(1,60) = 2.00 P = 0.163, lithium/CHIR99021 effect F(5,60) = 3.586 P = 0.03, Ank3 repression effect F(1,60) = 21.389 P < 0.001, LCM and lithium/CHIR99021 interaction F(2,60) = 7.505 P = 0.001. c Top: Representative western blot of α-tubulin in soluble (S) and polymerized (P) protein fractions. Bottom: Quantification of the ratio of soluble:polymerized tubulin. Univariate ANOVA, LCM effect F(1,60) = 17.561 P < 0.001, lithium/CHIR99021 effect F(5,60) = 0.203 P = 0.817, Ank3 repression effect F(1,60) = 68.479 P < 0.001, LCM and lithium/CHIR99021 interaction F(2,60) = 6.113 P = 0.004. Western blot data were averaged from two independent experiments with three biological replicates per group in each experiment. The data were analyzed using two-tailed Student’s t test or ANOVA and Bonferroni post hoc tests. Data are presented as mean ± s.e.m. *P < 0.05, **P < 0.01. n.s indicates not significant, C control sgRNA, A Ank3-targeting sgRNA