Figure 1: Expression of nELAVLs correlates with the APP695-specific pre-mRNA processing.

(A) High-throughput sequencing data from 17 human samples were analyzed for the association between APP isoforms and Hu, AUF-1 and TIA-1 mRNA expression. Color intensity and circle size indicate the strength of the correlation. Note that only APP695 is correlated with nELAVL expression. (B) Schematic representation of APP AS in neuronal and non-neuronal cells and localization of primers used in this study (arrows, F: forward, R: reverse) Boxes indicate exons and bold lines introns. RT-PCR was carried out using total RNA isolated from five cell lines and primary cortical neurons with primers specific for human or mouse ELAVL1, ELAVL2, ELAVL3, ELAVL4, and primers for human or mouse APP that allow the simultaneous detection of all AS events of exons 7 and 8. The indicated amplification bands resulting from AS of APP exons 7 and/or 8 were identified by their respective length. Quantification of the results was performed by scanning densitometry and the percentage of exclusion of both exons 7 and 8 is indicated below each lane. (C) Equal amounts of total protein from lysates of five cell lines and cortical neurons were analyzed on SDS-PAGE and immunoblotted with antibodies specific for ELAVLs, APP and GAPDH as a loading control. Note that similar to cortical neurons, significant levels of APP695 were observed only in the cells lines expressing nELAVLs. (D) Human SH-SY5Y and mouse CAD neuroblastoma cells were differentiated into a neuronal-like phenotype. Equal amounts of total protein from lysates of the above untreated and differentiated cells were analyzed on SDS-PAGE and immunoblotted with antibodies specific for the neuronal markers β-ΙΙΙ tubulin and SAP97 as well as for APP, ELAVLs and finally GAPDH as a loading control. Note that differentiated SH-SY5Y and CAD cells displayed a concurrent upregulation of nELAVLs and APP695.