TOE1 is recruited to Cajal bodies in a coilin-dependent manner. (A) Schematic representation of TOE1 mutants used in this study. ZnF, zinc finger domain; DEDD, deadenylation; FL, full length. (B) Mapping coilin-binding domain in TOE1. 293T cells were transfected with constructs encoding full-length or mutant TOE1. Pull-down experiments were performed using streptavidin beads and blotted with anti-Flag (for TOE1 mutants), anti-coilin, anti-DKC1, anti-FBL, or anti-SMN antibodies (please also see Fig. S4 A). (C) Coilin binding is a prerequisite for the loading of DKC1 and FBL, but not SMN, into TOE1-containing complexes. Constructs encoding TOE1 or a negative control NFYA were transfected into HeLa cells stably expressing control shRNA or coilin shRNA. Pull-down experiments were performed using streptavidin beads and blotted with anti-Flag (for negative control NYFA and TOE1), anticoilin, anti-DKC1, anti-FBL, or anti-SMN antibodies. Quantitative results showed the ratio (±SD) of the indicated proteins pulled down by TOE1 in coilin knockdown cells relative to those in control cells (n = 3 independent experiments). The asterisk indicates a nonspecific band. (D) TOE1 localizes to Cajal bodies in a coilin-dependent manner. Indicated mutants of TOE1 were transiently expressed in HeLa cells and then subjected to immunostaining using anti-Flag and anti-coilin antibodies. (E) Cajal body localization of TOE1 was abolished in coilin-depleted cells. Localization of TOE1 was detected in HeLa cells stably expressing control shRNA (top) or coilin shRNA (bottom) by immunostaining using anti-TOE1 and anti-coilin antibodies. (F) Quantitative results showed the percentage of cells (±SD) in which indicated proteins colocalized with coilin (n = 3 independent experiments). (G) Quantitative results showed the percentage of cells (±SD) expressing control or coilin shRNA in which TOE1 colocalized with coilin (n = 3 independent experiments). shCTL, control shRNA; WB, Western blot. Bars, 10 µm.