Research Paper Volume 11, Issue 23 pp 11268—11313

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.