FBXO44-Mediated Degradation of RGS2 Protein Uniquely Depends on a Cullin 4B/DDB1 Complex - Fig 1

<p>Protein expression of both RGS2 and RGS4 is dramatically increased by the proteasome inhibitor MG-132 as shown by Western blot (<b>A</b>) and the chemiluminescence-based PathHunter ProLabel assay (<b>B</b>). <b>C</b>. Co-immunoprecipitation of HEK-293 cells transiently transfected with RGS2 and HA-ubiquitin with or without treatment with 10μM MG-132. Proteasome inhibition results in a marked increase in protein levels and poly-ubiquitination of RGS2. <b>D</b>. The N-end rule pathway for the known substrate RGS4 and the hypothetical pathway for RGS2 (C; cysteine, G; glutamine; E; glutamic acid, R; arginine). <i>ATE-1</i> siRNA results in a significant increase in protein levels of RGS4, but has no effect on RGS2 protein levels by the PathHunter ProLabel assay (<b>E</b>) and Western blot (<b>F</b>). In both assays the protein levels of each RGS protein is compared to the levels with non-targeting siRNA (NT), but note that basal protein levels of RGS4-HA are much lower than RGS2-HA. Data are presented as mean ± S.D. of 3 independent experiments run in triplicate; **<i>P</i><0.01; ***<i>P</i><0.001 using one-way ANOVA with Bonferroni’s post hoc test for pairwise comparisons.