Figure 1. Analysis of GPR21 expression and activity.

Epididymal fat pads of control and HFHS-fed C57BL/6J mice were lysed, proteins separated by SDS-PAGE and expression of (a) GPR21 and (b) the macrophage marker, F4/80, evaluated by Western blotting. Representative Western blots are shown along with relative densities, determined using Image J software, presented as mean ± SEM, n = 9. Using the unpaired Student’s t-test a significant increase in F4/80 expression was observed at p < 0.01**. (c) Representative Coomassie stained gel used to confirm equal loading of protein extracted from murine epididymal fat pads. (d) A FRET-based IP-one assay was employed to assess the activity of GPR21 in transiently transfected HEK293T cells when coupled with the Gα_q G protein subtypes, Gα_q, Gα_15/16 or Gα₁₄. HEK293T cells were also transfected with the empty vector, pCMV6-Entry. Data presented as mean ± SEM are representative of three independent experiments performed in triplicate each time. One-way ANOVA with a post-hoc Tukey test denotes a significant increase in GPR21-induced IP₁ production at p < 0.05* and p < 0.001***. As Gα_q stimulation induces PLC activation to trigger the inositol cascade, the effect of the PLC inhibitor, U73122, on IP₁ production was also assessed in HEK293T cells transiently transfected with (e) pCMV6-Entry or GPR21 and (f) pCMV6-Entry or GPR21 coupled with Gα_15/16. GraphPad Prism^® 5 software was used to generate a non-linear sigmoidal dose response curve in response to U73122. Data presented as mean ± SEM are representative of two independent experiments performed in triplicate each time.