Research Paper Volume 11, Issue 23 pp 11268—11313

Figure 1. Discovery of RXFP3 as a possible controller for GIT2, identified through an expressional relationship. (A) Using transcriptome profiling, we investigated the expression levels of RXFP3 in GIT2-knock out (GIT2KO) mice (n=4). Both in the central nervous system (CNS) (cortex, hippocampus, and hypothalamus) and in peripheral tissues (pancreas, and liver) RXFP3 expression level was decreased compared to Wild-type (WT) littermates (n=4), GAPDH expression was shown to be stable as a control. (B) These results were replicated using western blotting, actin loading control was used. (C) Through transient transfection, RXFP3 was over expressed in SH-SY5Y and HEK293 cells at 1 and 2 μg, as control cells were also overexpressed with an empty vector (pcDNA3). Actin was used as a loading control (n=3). A specific expression increase for GIT2 was seen after overexpression of RXFP3 in both cell types. (D) Stimulation of GT1-7 cells using the endogenous ligand Relaxin 3 (RLN3), showed a dose dependent increase in GIT2 expression, with increasing levels of RLN3 (n=3). Data represent the means ± SEM (standard error of the mean). Statistical analyses (Student’s t-test) were performed using GraphPad Prism version 7.0 (GraphPad Software, San Diego, CA, USA). Significance level is indicated in each figure as *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001.