Figure 1

Hisactophilin is enriched in the cytoskeletal fraction of hyperosmotically shocked cells. (A) Hisactophilin is enriched in the cytoskeletal fraction of cells hyperosmotically shocked for 2 h with respect to control cells suspended in SPB buffer for 2 h. (upper frames). Correspondingly, less hisactophilin was found in the cytosol of cells exposed to hypertonicity when compared to cells suspended in SPB buffer (lower frames). Proteins from cytosolic and cytoskeletal fractions were separated by 2-D electrophoresis. The frames show a silver-stained region of the gel corresponding to a molecular weight of 15 kDa and to an IEP of 7.3–7.5. The predominant spot of each panel is indicated with an arrow. (B) Identification of the protein in (A) as hisactophilin by Western blotting of a typical gel of the cytoskeletal fraction and immunostaining of with a α-hisactophilin antibody. (C) The enrichment of hisactophilin in the cytoskeletal fraction of cells exposed to hypertonicity was confirmed by 1-D SDS-PAGE. The cytoskeletal fraction was isolated from cells suspended in SPB buffer (-) or in SPB buffer/400 mM sorbitol (+) for 2 h. The proteins were separated by SDS-PAGE and blotted. Immunostaining was performed with an α-hisactophilin antibody (right panel). The membrane was subsequently coomassie-stained (left panel). Abb.: M= marker.