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Fig. 2. GATA2 in differentiating ES cells and the generation of inducible ES
cells. (A) Rapid induction of Gata2 upon short-term
treatment with BMP4. ES cells were differentiated for 2 days in the absence of
serum, collected and treated with BMP4, or 10 µg/ml cycloheximide (CHX), or
DMSO for 1 hour. Gata2 expression was analyzed and normalized to
Gapdh. The quantity of Gata2 expressed in untreated cells
was normalized as 1 and used to determine the Gata2 quantity in
BMP4-treated samples. Values indicate mean±s.e.m.; *,
P<0.01 versus control. (B) Expression kinetics of candidate
genes in A2Lox ES cells. RNA from EBs differentiated in serum was utilized in
qRT-PCR assays. Candidate genes were first normalized against Gapdh
and then the maximal expression for each gene was assigned 100% and the
according value for each EB day determined against this 100% value. (C)
Schematic of the iGATA2 ES line used, with indicated loci carrying alterations
allowing for production of the rtTA, expression of the Gata2 cDNA and
generation of hCD4 as a surrogate marker for Scl. (D) Western
blot of the iGATA2 ES line. Cells were differentiated in serum for two days,
treated with the indicated concentrations of Dox and differentiated for two
additional days. EB lysates were subjected to SDS-PAGE followed by blotting
with GATA2 and ß-actin antibodies.
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