Determination of the half-life of the different glycosylation states of WT and Nramp2^G185R. (A) LLC-PK₁ cells stably transfected with either WT (top) or Nramp2^G185R-HA (bottom) were treated with cycloheximide (20 μg/mL) for the indicated periods of time. Whole-cell lysates were then subjected to electrophoresis and immunoblotting with anti-HA antibodies. (B) Rate of disappearance of the complex-glycosylated form of WT Nramp2 (▪) and Nramp2^G185R (○), from 4 experiments like that in panel A. (C) Cells were incubated overnight with 2.5 μg/mL tunicamycin before addition of cycloheximide. Analysis was done as for panel A. (D) Rate of disappearance of the core- and unglycosylated forms of WT Nramp2 and Nramp2^G185R, from 4 experiments like that in panel C. Data in B and D are means plus or minus SE.