Fig. 8

Activity dependent alternative splicing of Tpm1. a Tpm1 is encoded by 15 exons (yellow boxes, not to scale). Differential exon usage results in at least 21 splice variants. Of these, 4 are shown in green. Larger boxes represent coding sequence and smaller boxes untranslated sequence. b Visualization of the microarray data obtained for Tpm1. The plot shows the log2 ratio calculated from the mean normalized intensities of transformed expression values over the course of probesets per exon. Each green line represents expression kinetics of one time point after seizure onset. The blue boxes represent the probesets per exon present on the microarrays. c Validation of Tpm1 variant expression in hippocampus of control mice or 1, 4 or 8 h after seizure onset by RNA sequencing. Shown are coverage plots (blue) from paired-end reads for the four sample groups. The reference gene track is depicted below (black). d Sashimi plot of RNA sequencing data for Tpm1. Numbers indicate the counts of RNA sequencing reads that span the respective exon junctions. e Validation of splicing events by RT-PCR using indicated exon-spanning primer pairs (arrows). The respective exons are indicated by numbers. Primers were used to assess expression of exons in total RNA of hippocampi of control mice (−) and of mice sacrificed 4 h after onset of seizures (+). Note, forward primers corresponding to exon 1 and 4 match either to the 5′-untranslated sequence or to the coding sequence of the exon. The reverse primer matching exon 13 corresponds to the 3′ untranslated region and the revers primers corresponding to exon 14 match to the coding or the 3′ untranslated regions. The double bands produced by using primers corresponding to exon 11 and 13 indicate amplification of transcripts including (upper band) or excluding (lower band) exon 12