Loss of Grab leads to impaired endocytic recycling of Tfrc. (A) Representative fluorescence images of GRAB and the indicated organelle markers in differentiating K562 cells. Arrowheads indicate recycling endosomes. Scale bars, 10 μm. (B) Quantification of the colocalization between GRAB and the markers in differentiating K562 cells. Pearson's correlation coefficients are shown. N = 30 for each group. **P < .01 compared with the TFRC group. (C-D) Immunofluorescence assays of Tfrc and Rab11a in wild-type and Grab knockout MEL cells (C) and K562 cells (D). Arrowheads indicate recycling endosomes. Scale bars, 10 μm. (E-F) Immunoblotting analyses of Tfrc and Rab11a in wild-type and Grab-deficient MEL cells (E) and K562 cells (F). (G) Quantification of the Tfrc protein abundance in the control and Grab-silencing mouse primary fetal liver erythroblasts. *P < .05. (H-I) Flow cytometric analyses (H) and fluorescence assays (I) showing delayed clearance of internalized Tf-Alexa 488 in Grab knockout cells. Following the internalization of Tf-Alexa 488 for 2 minutes, the cells were transferred to fresh medium without Tf-Alexa 488 and analyzed at indicated time points. *P < .05. Scale bars, 10 μm.