CLIP-seq identifies mRNAs that bind DAP5. (A, top) Schematic representation of DAP5 domain organization. Locations of epitopes of the MBL and CS anti-DAP5 antibodies that were used for IP are indicated with black arrows. Red bar indicates the location of the epitope of the mouse monoclonal anti-DAP5 antibody that was used for western blotting. (Bottom) Western blot of total cell lysate (TCL) and DAP5 IP with the two rabbit anti-DAP5 antibodies. Rabbit IgG (RIgG) was used as a control. (B) Principal component analysis (PCA) of transcript profiles of the two IPs, each performed in triplicate, and total mRNA from the same experiments, showing the variability among the samples. The two components of the PCA explain 98% of the variance among the samples. (C) Volcano plots of DAP5 CLIP-seq results from RNA IP using CS antibody (left) and MBL antibody (right). Blue dots indicate mRNAs that were enriched in the DAP5 bound fraction compared to the total RNA (Fold-change > 2, FDR < 0.05). (D) Venn diagram comparing the overlap between the sets of DAP5 bound mRNAs identified in the two IPs. (E) Venn diagram comparing the overlap between the set of DAP5 bound mRNAs identified by CLIP and the set of DAP5 translationally activated mRNA targets identified by RP. The overlap was statistically significant, as determined by cumulative distribution function (CDF) of the hypergeometric distribution.
