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Fig. 1.
Interaction between NDC1 and ALADIN in vitro and in vivo. (A) Interaction of NDC1 with ALADIN in vitro. ALADIN was generated by invitro-translation and incubated with GST-NDC1 (amino acids 287–386) or GST bound to Sepharose. Bound proteins were analyzed by SDS–PAGE followed by autoradiography. (B) Interaction of NDC1 with ALADIN in vivo. Lysates prepared from HeLa 2-MeOE2 were immunoprecipitated with the indicated antibodies, separated by SDS–PAGE and analyzed by immunoblotting with the indicated antibodies. (+) Antibodies were pre-incubated with antigen before immunoprecipitation. (C) NDC1 and ALADIN are colocalized at the nuclear envelope in HeLa cells. Cells were cotransfected with FLAG-NDC1 and GFP-ALADIN and stained with anti-NDC1 antibody. Cell nuclei were counterstained with ToPro3. Scale bar, 5 μm.
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We next examined whether endogenous NDC1 is associated with ALADIN in vivo. Lysates from HeLa cells were subjected to immunoprecipitation with anti-NDC1 antibody followed by immunoblotting with anti-ALADIN antibody. NDC1 was identified as a 70 kDa protein and was found to coimmunoprecipitate with ALADIN ( Fig. 1B). Likewise, immunoprecipitation of the lysates with anti-ALADIN antibody followed by immunoblotting with anti-NDC1 antibody revealed an association between NDC1 and ALADIN. Coprecipitation of NDC1 and ALADIN was inhibited by preincubation of the antibodies with the antigens used for immunization.