Analysis of the degradation mechanisms of WT and Nramp2^G185R. LLC-PK₁ cells transfected with WT or Nramp2^G185R-HA were labeled metabolically by a one-hour pulse of ³⁵S-Met/Cys. Samples of the labeled cells were taken immediately after the pulse (0 chase) or after a 2-hour chase in the absence of presence of either 10 μM of MG132 or 15 mM of NH₄Cl, as indicated. The cells were next subjected to lysis, immunoprecipitation of Nramp2, gel electrophoresis, and autoradiography, as in Figure 4. (A) Typical radiograms of WT (top) and Nramp2^G185R (bottom) transfected cells. Only the band corresponding to the core-glycosylated species is shown. (B) Quantification of the fraction of core-glycosylated Nramp2 remaining after a 2-hour chase, normalized to the amount of the respective protein immediately after the radioactive pulse. Data are means plus or minus SE of 3 experiments like that shown in panel A. ^***P < .001, calculated using Student t test.