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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.
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