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Fig. 6. Overexpression of HDGF in mouse fetal gut explants retards epithelial
differentiation. (A) UV micrograph showing expression of GFP-tagged HDGF in a
sample E14 explant 18 hours after plasmid electroporation. (B) Immunoblot
confirming that full-length and mutant (m) HDGF were expressed to similar
levels. Duplicate samples are shown for each; the prominent faster-migrating
protein band represents a non-specific immunoblot signal and surrogate loading
control. (C) RT-PCR analysis of cultured intestinal explants for molecular
markers of gut epithelial differentiation after forced expression of GFP (CTL)
or GFP-tagged wild-type or mutant (m) HDGF. Explant RNA was isolated 1 and 2
days after transfection and analyzed by RT-PCR for transcript levels of
differentiation markers: apolipoprotein A1 (Apo1a), liver (Fabpl) and
intestinal (Fabpi) fatty acid-binding proteins, proline-rich acidic protein 1
(Prap1), villin and metallothionein 2 (Mt2). The results represent five
independent experiments with intact and two separate studies with the mutant
form of HDGF. Gapd, loading control. RT+ and RT– refer to mRNA samples
treated with and without reverse transcriptase, respectively.
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