Figure 3.

Expression of a C/EBP phosphorylation mutant at the Erk and Rsk sites inhibits neurogenesis in culture. a, Schematic of C/EBPβ wild-type protein. Two activation domains (ADI and ADII) are situated to the N terminus (NH₂), whereas the DNA-binding domain (DBD) and the leucine zipper dimerization domain (LZ) are located at the C terminus (COOH). The two phosphorylation sites investigated in this study, threonine-188 (T188; a substrate for ERK) and threonine-217 (T217; a substrate for Rsk), are also shown. b, Western blot analysis for the different C/EBPβ mutants expressed in HEK 293 cells showing expression of proteins of the appropriate sizes. c, Double-label immunocytochemical analysis for GFP (green) and βIII-tubulin (red) at 4 DIV on precursor cells that were transfected with either GFP plus the empty vector or GFP and the C/EBPβ T/A phosphorylation mutant. Arrows represent cells that coexpress both markers and arrowheads represent cells that were transfected but that do not express βIII-tubulin. Cells were counterstained with Hoechst (blue) to show all of the nuclei in the field. d, Quantitation of data similar to that shown in c for precursor cells transfected with GFP plus empty vector, GFP and the C/EBPβ T/A mutant, or GFP and the CA-C/EBPβ phosphorylation mimic, cultured for 4 DIV and immunostained for βIII-tubulin. ^*p < 0.01, relative to control-transfected cultures (ANOVA or Student's t test). e, Quantitation of data similar to that shown in c for precursor cells stimulated with CNTF after transfection with plasmids encoding GFP plus empty vector, GFP plus the C/EBP T/A mutant, or GFP plus A-C/EBP. Cells were immunostained for GFAP after 4 DIV. ^*p < 0.01, relative to control-transfected cultures (ANOVA or Student's t test). Scale bar, 100 μm. Error bars indicate SEM.