Fig. 1. Sox2 expression during in vitro neural stem cell differentiation. (A) In vitro neural stem cell differentiation scheme. (B) Specificity of the anti-Sox2 antibodies used in immunocytochemistry. Differentiation day 1 and 9 of wild-type (wt) and Sox2 conditionally deleted (null) cells are shown. Left, R&D antibody; right, Chemicon antibody (see also Fig. S1 in the supplementary material). A clear nuclear signal is visible in wild-type, but not in Sox2-null, cells. A slight cytoplasmic staining can be seen with the rabbit antibody (Chemicon) in wild-type and null cells, thus likely representing a nonspecific background. (C) Sox2 and nestin immunofluorescence on differentiation day 1. We used Chemicon's anti-Sox2 antibody, confirming with R&D antibody. (D) RT-PCR of Sox2 expression in undifferentiated neurospheres (Undiff. NSC), day 9 differentiated cells (diff. NSC) and P0 cortical cells. Top: cDNA dilutions from undifferentiated NSC (0.1, 0.25, 0.5, 1) allow an estimate of Sox2 expression levels in differentiated (diff. NSC) and cortical cells. Bottom: 18S RNA PCR, for normalization. (E) Western blot of Sox2 (R&D antibody) in normal (+/+) and mutant (MUT) undifferentiated neurospheres. Upper band: ubiquitous CP2 transcription factor (loading control). Sox2 protein in the mutant is 15-25% of normal by densitometry.