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.