Fig. 5. Requirement of GFAP phosphorylation at Ser8 for GFAP-14-3-3 interaction. (A) Schemes showing the N-terminal amino acid sequences of human and mouse GFAP (upper), and its substitution mutations (below). (B) Representative immunostaining images of GFAP mutants and 14-3-3 in transfected SW13 Cl2 cells. Scale bar, 10 µm. (C) The bar graphs show quantification of the colocalization of GFAP mutants and 14-3-3 in (B). In each experiment, transfected interphase cells (n=100) were recorded and the displayed data are representative of three independent experiments. *The differences between S8A and WT (wide type) values marked by asterisks are highly significant (P<0.001). The differences between other mutants and WT values showed no significance (P>0.05). (D) The immunoprecipitation assay of GFAP and its serine site mutants with 14-3-3. Extracts from mutant transfected SW13 Cl2 cells were subjected to immunoprecipitation with rabbit anti-GFAP antibody, and subsequent western blot with mouse anti-14-3-3 and anti-c-myc antibodies. Whole cell lysates were also detected by mouse 14-3-3 antibody and rabbit GFAP antibody as the control.