Glycosylation, subcellular localization, and functional analysis of mutant G-CSFR^Arg308Cys. (A) Western blot of G-CSFR in HeLa cells expressing either WT or mutant G-CSFR^Arg308Cys (Mut) fused to a C-terminal eGFP. (B) Western blot upon treatment with N-glycosidases EndoH and PNGase F. (C) Confocal microscopy visualizing G-CSFR (eGFP) and ER (Calnexin). Localization of WT-eGFP and mutant G-CSFR^Arg308Cys-eGFP was imaged with a ×63 objective and 2.1 and ×2.7 zoom, respectively. (D) Flow cytometric analysis of cell surface expression of WT G-CSFR and mutant G-CSFR^Arg308Cys C-terminal eGFP fusion proteins. Comparison of eGFP and G-CSFR–PE signals reveals a linear relationship in WT, but not in mutant G-CSFR^Arg308Cys. (E) Western blot of HeLa cells expressing WT-eGFP or Mut-eGFP after incubation with 100 ng/µL rhG-CSF. Empty pMMP vector (E) was used as a control (A,E).