Figure 4.

TMEM187 deficiency increases iron uptake efficiency through the RAB11A-GRAB-TfR1 pathway in K562 cells. (A) The enhanced colocalization between endogenous GRAB (green) and TfR1 (red) in K562 KO cells before and after 4-day induction, revealed by immunofluorescence assays. (B) Representative fluorescence images showing FITC-labeled holo-transferrin uptake in WT and KO cells. Arrows indicate the magnified cells. Green, FITC-labeled holo-transferrin; blue, DAPI. (C) Protein levels of GRAB and RAB11A, quantified in (D). GAPDH was used as an internal control. One-way ANOVA was used for significance. n = 3/group. ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. (E-F) Representative fluorescence images of TMEM187-EGFP and GRAB (E), and TMEM187-EGFP and RAB11A (F). Red, endogenous GRAB or RAB11A; blue, DAPI. Scale bars, 10 μm. (G) Co-IP assays showing an interaction between EGFP-RAB11A and HA-TMEM187 in HEK293 cells. (H) Co-IP assays showing an interaction between 3×FLAG-RAB11A and HA-TMEM187 in K562 cells. (I-J) Representative fluorescence images of TMEM187-mcherry and the constitutively GTP-locked active form (RAB11A Q70L) (I) or GDP-locked inactive form (RAB11A S25N) (J) of EGFP-RAB11A in HEK293 cells. Red, TMEM187-mcherry; blue, DAPI. Scale bars, 10 μm. (K-L) The preference of interaction between TMEM187-HA and active/inactive form of EGFP-RAB11A (K) or between GRAB-HA and active/inactive form of EGFP-RAB11A (L), respectively, in HEK293 cells. (M) Interaction competition between EGFP-TMEM187:3×FLAG-RAB11A and GRAB-HA:3×FLAG-RAB11A in HEK293 cells, demonstrated by Co-IP assays. #, nonspecific band.