Fig. 4. Identification of the 14-3-3 interaction domain of GFAP. (A) Schematic presentation of GFAP and its deletion mutants. Like all IF proteins, GFAP contains an N-terminal head domain, a C-terminal tail domain and a conserved -helical rod domain. Two deletion mutants (H and T) of GFAP were constructed by PCR, and c-myc was inserted into the C-terminal of the mutants. (B) The immunoprecipitation assay of c-myc tagged GFAP and its deletion mutants with 14-3-3. SW13 Cl2 cell extracts were subjected to immunoprecipitation with rabbit anti-GFAP antibody, subsequent western blot with the mouse anti-c-myc (upper) and anti-14-3-3 (middle) antibodies. Whole cell lysates were also subjected to western blot by 14-3-3 antibody as the control (bottom). (C) Colocalization of GFAP and 14-3-3. SW13 Cl2 cells were transfected with GFAP and its mutants (H and T). The cells were immunostained by mouse anti-c-myc antibody (green) and rabbit anti-14-3-3 antibody (red). (D) FRET between 14-3-3-CFP and GFAP-YFP in transfected SW13 Cl2 cells. The fluorescent images showed the recordings immediately before and after the photobleaching cycle. The bleaching zone (ROI1) was marked with a red arrow and the control zone (ROI2) was marked with a white arrow. (E) The plot shows the fluorescence intensities in the two regions for each channel in Fig. 4D. (F) FRET efficiencies (Ef) between 14-3-3-CFP and YFP-tagged GFAP deletion mutants. Ef is represented as the differences of CFP fluorescence before and after YFP photobleaching in defined regions (ROI 1, see Fig. 4D). Similar calculations were done in a non-bleached region (ROI 2) of comparable intensity of the same cell to calculate the control value (Cf). In each experiment, n=10 cells were recorded and analyzed per condition. *The differences between the paired Ef and Cf values marked by asterisks are highly significant (P<0.001). The differences between the paired Ef and Cf values marked by # were not significant (P>0.05). Scale bar, 10 µm. Scale bar in magnified figure, 5 µm.