Fig. 1. Association of 14-3-3 and GFAP is independent of vimentin. (A) Immunoprecipitation assay by GFAP antibody in astrocytes. The astrocyte extracts (lane 1) were subjected to immunoprecipitation with a rabbit preimmune IgG (lane 2) or a rabbit anti-GFAP antibody (lane 3) and subsequent western blot analysis with mouse anti-vimentin antibody (top) or mouse anti-14-3-3 antibody (bottom). The pellet was also detected by vimentin and 14-3-3 antibodies (lane 4). (B) Immunoprecipitation assay by vimentin antibody in astrocytes. The astrocyte extracts (lane 1) were subjected to immunoprecipitation with a mus preimmune IgG (lane 2) or a mus anti-vimentin antibody (lane 3) and subsequent western blot analysis with rabbit anti-GFAP antibody (top) or mouse anti-14-3-3 antibody (bottom). The pellet was also detected by GFAP and 14-3-3 antibodies (lane 4). (C) Western blot analysis of SW13 Cl1 (lane Cl1) and SW13 Cl2 (lane Cl2) using specific antibodies to vimentin (top) and 14-3-3 (bottom). (D) Immunoprecipitation of 14-3-3 and vimentin. The extracts of SW13 Cl1 cells (lane 1) were subjected to immunoprecipitation with a rabbit preimmune IgG (lane 2) or the rabbit anti-14-3-3 antibody (lane 3) and subsequently detected with mouse anti-vimentin antibody (top) or mouse anti-14-3-3 antibody (bottom). The pellet was also subjected to western blot (lane 4). (E) Results of western blot of pcDNA3.1-GFAP transfected SW13 Cl2 cells. The proteins were detected by mouse anti-GFAP antibody (top) or mouse anti-14-3-3 antibody (bottom). The same amount of untransfected cells was used as in control (lane control). (F) The interaction of GFAP and 14-3-3 in pcDNA3.1-GFAP transfected SW13 Cl2 cells. SW13 Cl2 extracts transiently expressing GFAP (lane 1) were subjected to immunoprecipitation with rabbit anti-GFAP antibody (lane 3) or a rabbit preimmune IgG (lane 2) and subsequent western blot with mouse anti-GFAP antibody (top) or mouse anti-14-3-3 antibody (bottom). The pellet was also subjected to western blot analysis (lane 4).