DMT1 transport activity is decreased by WWP2 in an Ndfip-dependent manner that is reliant on the WW domain–PY motif interaction. (A) The relative transport activity of DMT1 in the presence of WWP2 (wild-type and catalytically inactive mutant, cm), Ndfip1 (wild-type and double PY mutant), and Ndfip2 (wild-type and double PY mutant) as indicated. *Significant difference between transfected and control CHO-DMT1-II cells (P < .05). **Significant increase (P < .05) from CHO-DMT1-II cells transfected with WWP2 and wild-type Ndfip1. ***Significant increase (P < .05) from CHO-DMT1-II cells transfected with WWP2 and wild-type Ndfip2. □ indicates CHO cells not expressing DMT1-II; ■, CHO-DMT1-II cells. Data are mean plus or minus SEM. (B) Immunofluorescence of Myc-DMT1 in untreated CHO-DMT1-II cells or after 4-hour incubation with MG132 (50 μM) or chloroquine (400 μM) as indicated. (C) Total levels of DMT1 in cells transfected with WWP2 and Ndfip1 or Ndfip2 and incubated for 8 hours with MG132 or chloroquine as indicated. DMT1 was immunoprecipitated using α-myc followed by immunoblotting. (D) The relative transport activity of DMT1 in the presence of WWP2 and Ndfip1 or Ndfip2 as indicated followed by incubation with MG132 or chloroquine for 8 hours as indicated. *Significant difference between transfected and control CHO-DMT1-II cells (P < .05). **Significant increase (P < .05) from CHO-DMT1-II cells transfected with WWP2 and Ndfip1 in the absence of inhibitors. ***Significant increase (P < .05) from CHO-DMT1-II cells transfected with WWP2 and Ndfip2 in the absence of inhibitors. □ indicates CHO cells not expressing DMT1-II; ■, CHO-DMT1-II cells. Data are mean plus or minus SEM.