Figure 4.

GPR37 protein expression is reduced upon knockdown of DENR or MCTS1. (A) GPR37 protein expression is reduced upon knockdown of DENR or MCTS1. The GPR37 transcript including 5′UTR, open reading frame, and 3′UTR, was cloned downstream of a constitutive CMV promoter, and co-transfected into HeLa cells together with a GFP expression construct bearing the same CMV promoter as a normalization control. GPR37 and GFP proteins were detected by immunoblots following the indicated siRNA-mediated knockdowns. Immunoblot is cropped for presentation purposes. Uncropped version in Supplementary Fig. S6. (B) Endogenous GPR37 mRNA shifts into lighter polysome fractions in PC3 cells upon DENR knockdown. (Top) Polysome profiles of PC3 cells treated with control or DENR siRNA. (Bottom left) Control immunoblot confirms DENR knockdown was efficient. (Bottom right) Quantitative RT-PCR on indicated fractions from the polysome gradient for endogenous GPR37 mRNA, normalized to hRpL19 mRNA, reveals that upon DENR knockdown GPR37 mRNA shifts into lighter fractions that are less translated. Error Bars: Std. dev. Student ttest ¹p ≤ 0.05, ³p ≤ 0.001 relative to ctrl RNAi.