Grab regulates the recycling of Tfrc through Rab8a. (A) Schematic of protein purification and mass spectrometry to identify Grab-interacting proteins. (B) Validation of the interaction between FLAG-Grab and Rab8a in MEL cells by coimmunoprecipitation assays. *Indicates immunoglobulin G (IgG). (C) Coimmunoprecipitation assays showing stronger interaction between HA-Rab8a (T22N) and FLAG-Grab in HEK293 cells. Rab8a (Q67L) and Rab8a (T22N) are constitutively GTP-locked and GDP-locked mutant forms of Rab8a, respectively. (D-E) Representative fluorescence images of Rab8a and Tfrc in wild-type and Grab-deficient MEL cells (D) and K562 cells (E). Arrowheads indicate recycling endosomes. Scale bars, 10 μm. (F) Quantification of the colocalization between Rab8a and Tfrc in wild-type and Grab knockout cells. Pearson's correlation coefficients are shown. The numbers of cells analyzed are 23 (wild-type) and 21 (both Grab knockout clones) for MEL and 30 for each of the K562 clones. **P < .01. (G) Immunoblotting analyses of Rab8a and Tfrc in wild-type and Rab8a knockout MEL cells. (H) Quantification of heme content in wild-type and Rab8a-deficient MEL cells following DMSO-induced erythroid-like differentiation. *P < .05. (I) Immunofluorescence analyses of Tfrc in Grab knockout MEL cells transfected with the wild-type (HA-Rab8a) or the constitutively active form (HA-Rab8a [Q67L]) of Rab8a. Arrowheads indicate recycling endosomes. Scale bars, 10 μm. (J) Total heme content in control zebrafish embryos, rab3il1 morphants, and rab3il1 morphants coinjected with rab8a (Q67L) mRNA. The zebrafish embryos were analyzed at 72 hours postfertilization. *P < .05; **P < .01. IB, immunoblotting; IP, immunoprecipitation.