Figure 7: ELAVL4 protein interferes with the binding of essential splicing factor U2AF65 upstream of APP exons 7 and 8.

Human SK-N-SH cells were co-transfected with two expression plasmids carrying either no insert (empty) or ELAVL4 in ratios depicted above each lane. (A) In order to ensure the presence of different ELAVL4 protein levels among the three conditions, equal amounts of total protein from lysates of the transfected SK-N-SH cells (input) were analyzed on SDS-PAGE and immunoblotted with antibodies against ELAVLs and β-TUBULIN (TUBB) as a loading control; membranes were also probed against U2AF65, to verify that ELAVL4 overexpression does not alter U2AF65 expression. (B) Whole cell lysates from the same transfected cells were used in a series of pull down assay using streptavidin beads and biotinylated RNA probes transcribed from the indicated human minigenes. The presence of U2AF65 and ELAVLs in the precipitates was assayed by immunoblotting. Quantification of the results was performed by scanning densitometry and the percentage (mean ± standard deviation) change of U2AF65-binding upon ELAVL4 overexpression is shown below each lane. Note, that ELAVL4-binding to these transcripts resulted in a reduction of U2AF65 binding (***P < 0.001).