Figure 2. 5 μg of RXFP3 overexpression indicates a role in the DNA damage response. (A) Western blot validation of the differential overexpression curve for RXFP3-HA (n=3). We see a clear increase of RXFP3-HA signal with an increased level of transfections. (B) We then selected the proteins unique to each overexpression level using a InteractiVenn for further investigation, obtaining the following percentages of uniquely-regulated proteins: 0.5 μg RXFP3 21.8% unique; 1 μg RXFP3 – 37.2% unique; 2 μg RXFP3 26.6% unique; 5 μg RXFP3 25.2% unique; 10 μg RXFP3 40.8% unique. (C) Applying protein-protein interaction (PPI) pattern analysis through Enrichr, we were able to show a strong representation for DNA damage repair and energy metabolism-related proteins across the different RXFP3 expression ranges. We also observed a strongest PPI dataset enrichment for DNA damage response proteins for the 5 μg RXFP3 expression level. (D–E) We next calculated the number of input dataset proteins associated with the target PPI database protein multiplied by the negative log10 of the enrichment probability, or hybrid score. We see that for the (D) sum and the (E) total sum of hybrid score occurrences, the enrichment for DDR-associated factors was shown to be most profound for the 5 μg RXFP3 level.