Detection of exofacial Nramp2 by biotinylation. LLC-PK1 cells stably transfected with either WT or Nramp2G185R-HA were treated with EZ-NHS-sulfoBiotin, a membrane-impermeant biotinylating reagent, as described in “Materials and methods.” Cell lysates were prepared and biotinylated membrane proteins were precipitated using mono-avidin beads. The biotinylated proteins were eluted, and analyzed by SDS-PAGE together with samples of the initial whole-cell lysate, transferred onto polyvinylidene difluoride (PVDF) membrane and blotted with anti-HA antibodies to identify Nramp2. The volume of whole-cell lysate (labeled “Total extract” in the figure) was equivalent to one tenth of the amount used for avidin precipitation in the corresponding lanes labeled “Biotinylated.” The blot is representative of 3 experiments.
