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Fig. 2 Necdin interacted with MYL12A (A) Interaction of necdin and MYL12A as assessed with a yeast two-hybrid assay. The term pGBKT7-necdin denotes necdin fused to a DNA-binding domain, whereas pGADT7-Myl12a denotes Myl12a fused to an activation domain. Only yeast colonies co-transformed with pGADT7-Myl12a and pGBKT7-necdin could grow and turn blue on an SD/-Leu/-Trp/AbA/X-?-Gal plate. (B) Interaction of necdin and MYL12A as assessed with a co-immunoprecipitation (coIP) assay. H9C2 cells were transfected with the indicated plasmids, and protein lysates were precipitated with antibodies against HA or Flag. Immune complexes were blotted with antibody against HA or Flag, respectively. (C) Necdin interacts with MYL12A endogenously as assayed with a coIP assay in H9C2 cells. Cell lysates were immunoprecipitated with antibodies against necdin, MYL12A, or control IgG, and immune complexes were blotted with antibodies against necdin or MYL12A, respectively. (D) Interaction of MYL12A and full-length or truncated necdin as assayed by a coIP assay. HA-tagged MYL12A and FLAG-tagged necdin fragments were expressed in H9C2 cells; the cell lysate was immunoprecipitated with Flag antibody, and the immune complex was blotted with HA antibody and Flag antibody, respectively.