1. Interactions between Aiolos and Ikaros proteins are detected within the cell nucleus.
Interactions between Aiolos and Ikaros proteins are detected within the cell nucleus. (A) Aiolos and Ikaros form complexes in lymphocytes. Nuclear extracts prepared from primary lymphocytes were immunoprecipitated with an affinity‐purified antibody to Aiolos which does not cross‐react with Ikaros (see Figure 1B). The immunoprecipitate was fractionated on an SDS‐acrylamide gel and detected with an antibody to Ikaros. The total nuclear extract contains three bands which react to the Ikaros antibody (lane 1). Two of these bands, corresponding to the 57 and 48 kDa Ikaros isoforms are co‐precipitated by the Aiolos antibody. This interaction is also observed in transfected cells. Aiolos–(Flag) and Ikaros proteins expressed in the epithelial cell line 293T were immunoprecipitated using an antibody to the Flag epitope. Immunoprecipitates were run on a 10% SDS gel and analyzed by Western blotting with an Ikaros antibody (lanes 3–5). The positions of Ikaros and Aiolos–Flag are indicated by the upper and lower arrows, respectively. Ikaros–Aiolos complexes were immunoprecipitated by the Flag antibody (lane 3), but the dimerization mutant IkM (Sun et al., 1996) was not precipitated when co‐expressed with Aiolos (lane 4). No Ikaros was observed in immunoprecipitates from untransfected controls (lane 5). To confirm the levels of Ikaros and Aiolos protein produced in the transfected cells, Western analyses on total protein were performed with the Ikaros (lanes 6–8) and Flag (lanes 9–11) antibodies. Similar amounts of Ik‐1 (lane 6) or IkM (lane 7) and Aiolos proteins (lanes 9 and 10) were produced in both transfected populations. No immunoreactivity was observed in untransfected cells (lanes 8 and 11). These results confirm that Aiolos and Ikaros form stable heterodimers in solution via their C‐terminal zinc finger motifs. (B) NIH 3T3 fibroblasts were transfected with Aiolos–Flag, Ik‐1 and Ik‐6 expression vectors either alone or together. Cells were stained with anti‐Ikaros and with anti‐Flag antibodies (Sun et al., 1996). When transfected alone, Aiolos and Ik‐1 proteins show punctate nuclear staining (panels 1 and 2) while Ik‐6 is detected throughout the cytoplasm (panel 3). When Aiolos is co‐transfected with Ik‐6, the Ik‐6 protein is found in the nucleus (panel 4). Note that in cells transfected with both proteins (Ik‐1 and Aiolos) Ikaros detected with FITC‐conjugated secondary antibody and Aiolos detected with rhodamine‐conjugated secondary antibody are found in similar locations within the nucleus (panels 5 and 7). When superimposed, the red and green signals generate a yellow signal confirming the co‐localization of these proteins (panel 6). Endogenous Ikaros detected in thymocytes also displays this punctate pattern of nuclear staining (panel 8), as does endogenous Aiolos (panel 9).
Bruce Morgan et al. EMBO J. 1997;16:
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