(A) Schematic illustration of the tBDNF protein coding sequence (upper panel) showing demethylated CG sites during conditioning in red, demethylated CA sites in yellow, and methylated CA sites in purple. The region deleted by splicing is indicted by the green box. The relative coverage of primer pairs P1-P3 used for ChIP-qPCR is indicated. The middle panel shows the methylation status of the tBDNF coding sequence determined by bisulfite sequencing PCR (BSP) in naïve (N), pseudoconditioned (Ps), and conditioned preparations (C15 min). Nearly all CG sites are significantly demethylated after conditioning for 15 min. 3 × 7 clones/group. As shown in the lower panel, specific CA sites also undergo conditioning-related methylation or demethylation. Bars in white are sites that are methylated in naïve and completely demethylated to 0% in conditioning; bars in black are unmethylated in naïve and methylated in conditioning. 3 × 10 clones/group. Red, green, and yellow lines indicate coverage by primer sets P1-P3, respectively. #p<0.05; *p<0.01. (B) Expression of the spliced tBDNF2a transcript is significantly inhibited relative to naïve by the DNMT inhibitors zebularine (Zeb; 100 µM) or RG108 (RG; 200 ng/µl) applied to the bath as shown by the semi-quantitative analysis. Levels of tBDNF2a transcripts from pseudoconditioned (Ps1) or conditioned (C1) preparations for one pairing session are also shown. (C) ChIP-qPCR assays for total MeCP2 protein show that primer set P1 detects MeCP2 tightly bound to tBDNF in naïve preparations during splicing of tBDNF2a (2aON) and is released after conditioning when splicing stops and tBDNF2a is suppressed (2aOFF). Application of the MeCP2 siRNA to naïve preparations results in significantly reduced ChIP signal in the P1 region compared to normal naïve. (D) Western blots confirm that levels of total MeCP2 are not altered during conditioning.