Fig. 4.

Effect of blocking Stat3 function on astrocytic differentiation. a, Cells were transiently transfected by the calcium phosphate method with plasmids expressing either wild-type (WT) or dominant negative mutant of Stat3 defective for DNA binding (mutant), both proteins expressing an N-terminal FLAG epitope. Forty-eight hours after transfection cells were treated with CNTF for 48 hr and fixed with freshly mixed methanol and acetone in a 1:1 ratio for 2 min at room temperature, and double-immunofluorescent staining was performed for FLAG and GFAP. A–C, Representative experiment of cells transfected with the mutant Stat3. D–F, Representative experiment of cells transfected with WT Stat3. A, D show staining specific for the FLAG epitope on the transfected proteins,B, E for GFAP, and C, F for both antigens. b, Based on experiments performed as described in Figure 3a, the percentage of transfected cells expressing GFAP was plotted for each of the two plasmids. Blocking of Stat3 function with a dominant negative protein inhibits CNTF-mediated astrocytic differentiation.