Fig. 6. Ankrd11 knockdown induces abnormal BDNF/TrkB signaling.

(A) Ankrd11 knockdown decreases the levels of bdnf and Trkb mRNAs. E16.5 cortical neurons were cultured and infected with control or shAnkrd11 lentivirus. RT-PCR was performed using total RNAs from cellular lysates (B) Quantification of mRNA levels shown in (A). The relative mRNA levels were normalized to Gapdh expression. N= 3 independent cortical cultures using 3 mice. Statistical significance was determined by two-tailed Student’s t-test. *p < 0.05, **p < 0.01. (C) Ankrd11 knockdown decreases the phosphorylation levels of BDNF/TrkB signal transducers. Western blots shows phosphorylation of AKT, GSK-3, S6K, and S6 in control and ANKRD11-deficient neurons. (D) Quantification of (C). The protein levels were normalized to total protein levels. N= 3 independent cultures using 3 mice. Statistical significance was determined by two-tailed Student’s t-test. *p < 0.05, **p < 0.01. (E) Levels of epigenetic regulators on the Trkb promoter. ChIP assays were performed using neuronal lysates and an antibody to ANKRD11, acetyl-Histone H3, acetyl-p53, MeCP2, or DNMT1, followed by PCR to measure the levels of the Trkb promoter associated with the epigenetic proteins. (F) Quantification of (E). The relative levels were normalized to the input fraction of the acetyl-Histone H3. N= 3 independent cortical cultures using 3 mice. Statistical significance was determined by two-tailed Student’s t-test. **p < 0.01, ***p < 0.001. (G) Levels of epigenetic regulators on the CpG region of the Trkb exon II. ChIP assays were performed and the CpG region was amplified using PCR. (H) Quantification of (G). The relative levels were normalized to the input fraction of acetyl-Histone H3. N= 3 independent cortical cultures using 3 mice. Statistical significance was determined by two-tailed Student’s t-test. ***p < 0.001.