Fig. 3.

An intact amino-terminal RCC1-like domain is required for correct subcellular localization. (A) Schematic representation of the disease-associated ALS2 mutant and amino-terminal deletion expression constructs. Expression was verified by immunoblotting transiently transfected COS cell lysates as indicated. DH, Dbl homology; PH, pleckstrin homology; MORN, membrane occupation and recognition nexus. (B) Representative deconvolved micrographs of COS cells transiently transfected with indicated constructs and stained with pAb-ALS2_RCC1 and FITC-conjugated goat anti-rabbit IgG. (C–I) Subcellular localization of ALS2 depends on an intact amino-terminal RCC1 domain. (C) HEK293 cells transiently transfected with amino-terminal-deleted ALS2 mutants were fractionated (see scheme in Fig. 1F) and immunoblotted with anti-FLAG M2 antibody. (D–I) Representative deconvolved images of COS cells transiently transfected with indicated constructs and stained with FITC-conjugated anti-FLAG M2 antibody (green, D–I) and endogenous (red) EEA1 (H) or transferrin receptor (TfR) (I). (Magnifications: ×600.)