Figure 1.

MISSL is a bona fide ALG-2-interacting protein. A, HeLa cells were transiently transfected with plasmids for expression of GFP or GFP-MISSL, and cell lysates were subjected to immunoprecipitation (IP) with an anti-GFP antibody in the absence or presence of 5 mm EGTA (+EGTA). The immunocomplexes were analyzed by immunoblotting with the indicated antibodies. B, HeLa cell lysate was subjected to IP with an anti-MISSL antibody (sc-243408) or control (ctrl) IgG in the presence of 5 mm EGTA or 100 μm CaCl₂. The immunocomplexes were analyzed by immunoblotting with the indicated antibodies. C, schematic representation of MISSL structure. Two putative ABM-1-like sequences, which are located at 101–117 and 167–175 amino acids (a.a.), are shown. The deletion mutants used are also shown below the MISSL structure. D, HeLa cells were transiently transfected with GFP and GFP-tagged full-length MISSL (FL) GFP-MISSL lacking both 101–117 and 167–175 amino acids (ΔΔ), and the cell lysates were subjected to IP with the anti-GFP antibody. The immunocomplexes were analyzed by immunoblotting with the indicated antibodies. E, HEK293T cells transfected with the plasmids for expression of the indicated proteins were lysed, and GFP or GFP-MISSL variants were immunopurified using the anti-GFP antibody. The immunoprecipitates were separated by SDS-PAGE and subjected to far-Western (FW) analysis using biotin-labeled ALG-2 (biotin-ALG-2) and to Western blotting (WB) with the anti-GFP antibody. F, IP analyses using HeLa cells transiently expressing GFP, GFP-MISSL full-length (FL), or GFP-MISSL deletions were performed as in D.