Figure 6
From: CLIMP-63 is a gentamicin-binding protein that is involved in drug-induced cytotoxicity
CLIMP-63 binds to 14-3-3θ, which in turn binds to 14-3-3β. (a) Immunoprecipitation was conducted on cell lysate from FLAG-tagged CLIMP-63 (FLAG-p63)-transfected 293T cells, using anti-FLAG antibody. SDS-gel electrophoresis followed by silver staining revealed a band specific to cells treated with 5 mM gentamicin for 24 h (+GT), which was not present in no gentamicin control (−GT), as indicated by an arrow. Mass spectrometry on the band identified 14–3–3β and θ. FLAG-p63 band is indicated by an arrowhead. The band was white because of the silver staining saturation effect. (b) 293T cells were co-transfected with FLAG-p63 and Myc-tagged 14–3–3β or θ (Myc-14–3–3β or θ), and immunoprecipitation using anti-FLAG antibody was performed after 24 h of incubation. FLAG-p63 was co-immunoprecipitated with Myc-14–3–3θ, but not β. (c) GST fusion pull-down assay was performed on cell lysate from Myc-14-3-3θ-transfected 293T cells. Coomassie blue staining of GST fusions followed by SDS-gel electrophoresis confirmed that comparable levels of GST fusions were used (left panel). Western blotting on the pull-down samples showed that Myc-14-3-3θ binds only to the cytosolic domain of CLIMP-63 (p63-Cyto), and not to GST or the lumenal domain (p63-lumen). (d) FLAG-tagged GFP or 14-3-(FLAG-GFP or 14-3-3θ) was co-transfected with Myc-14-3-3β. Immunoprecipitation using anti-FLAG antibody also pulled down Myc-14-3-3β, suggesting that 14-3-3θ and β binds with each other, probably forming a dimer. (e) COS-7 cells were co-transfected with CLIMP-63 and Myc-14–3–3θ or β. In the absence of exogenous CLIMP-63 expression, Myc-14-3-3θ was localized in the cytoplasm, and co-localization with endogenous CLIMP-63 was not very clear (upper panels). CLIMP-63 overexpression altered localizations of not only CLIMP-63 but also Myc-14-3-3θ, more distributed along peripheral sites of cells (middle panels). There was also significant co-localization between CLIMP-63 and Myc-14–3–3θ, as indicated by arrows. Myc-14-3-3β did not show clear co-localization with overexpressed CLIMP-63, but still altered localization, possibly through endogenous 14-3-3θ protein (lower panels)
