PKCα decreases endocytic membrane targeting and increases exocytotic membrane targeting of Fpn. (A) The protein expression of Fpn and FTH1 was determined in BMDMs from WT and PKCα^−/− mice. (B) Fpn mRNA expression was determined in WT and PKCα^−/− BMDMs. (C) WT BMDMs were treated for 4 or 24 hours with 5 μM Go6976 or 2 μM Go6983, followed by biotin/streptavidin pulldown of cell surface proteins. The total and surface Fpn were then determined by blotting with anti-mouse Fpn antibody. (D) TRex cells were treated overnight with Dox (100 ng/mL) to induce Fpn-GFP expression, followed by a 2-hour pretreatment with cycloheximide (CHX, 100 μg/mL), an inhibitor of de novo protein synthesis, before treating with Go6976 or Go6983 for 4 hours. Subcellular localization of Fpn-GFP was then assessed by confocal microscopy. Red arrow denotes intracellular Fpn-GFP. (E) TRex cells were transduced with PKCα-CAT, or pretreated with Go6976 or Go6983, before a 4-hour treatment with Dox (1 μg/mL) and confocal imaging. (F) TRex cells transfected with backbone (BB) or PKCα-CAT were treated with Dox (1 μg/mL) for 0.5, 2 or 4 hours. Surface biotinylation was performed, and the total and surface Fpn-GFP expression was determined by blotting with anti-mouse GFP antibody. (G) TRex cells were treated as indicated underneath the blots. Both sets of cells were subjected to surface biotinylation at the end of 4-hour treatment by Go6983 or the control. Data shown are mean ± SEM of 3 independent experiments. ∗P < .01 compared with WT or the control conditions. ^#P < .05. Bar, 10 μm.