TMD4 mutations in Nramp2 do not alter localization to the plasma membrane, but result in decreased protein levels. Epitope-tagged wild-type, G185R, G184D, and D192G Nramp2 cDNA mutant constructs were transfected into HEK 293T cells, and cells were treated as described in Fig 3B. To quantitate the reduction in mutant protein levels, the cell lysate from cells transfected with wild-typeNramp2 was diluted 1:4, 1:10, and 1:40 in RIPA buffer. The amount of undiluted protein (lane 2) and diluted protein (lanes 3 to 5) was then compared with the amount of undiluted mutant protein detected (lanes 7 to 9). The antisense (-) control is shown in lane 6. Sizes of molecular weight standards (lane 1) are indicated on the left.