Figure 3.

The prevention of N-glycosylation leads to reduced cell surface expression of Gpr176. (A) Representative confocal images of Flp-In TREx293 cells expressing WT (upper) or N-ter mut Gpr176-GFP (lower). Nuclei were stained with DAPI. The merged image is a combined image of GFP (green) and DAPI (blue). Gamma levels were adjusted over the whole image to optimize the appropriate GFP signals. The images are representative of three experiments. Scale bars, 10 μm. (B) Confocal images showing intracellular accumulation of N-ter mut Gpr176-GFP (green) in ER. Cells were stained with ER-Tracker (red). The merged image is a combined image with DAPI (blue). Cells are representative of a population with independent experiments repeated four times. Scale bars, 10 μm. (C,D) Immunoblots examining the effect of MG132 (C) and bafilomycin A1 (D) treatment on the expression of WT and N-ter mut Gpr176-GFP. Cells were treated with MG132 (C) or bafilomycin A1 (D) at the indicated concentrations for 6 h. The protein extracts of N-ter mutant cells were loaded at five-fold higher levels compared to those of WT cells to increase the sensitivity of protein detection. Note that mutant expression was partially restored by MG132 (C) but not by bafilomycin A1 (D), while under both conditions, WT expression was nearly unchanged.