FIGURE 2.

Validation of GIIIc reporters in vitro. (A) Western blots indicate that rat DT cells differentially express epithelial markers E-cadherin and pan-cytokeratin, and rat AT3 cells express mesenchymal markers fibronectin and vimetin. β-actin was used as a loading control. (B) RT-PCR and restriction digestion reveal that DT and AT3 cells express the endogenous FGFR2 IIIb and IIIc isoforms, respectively. (M) Mock digested RT-PCR amplicon; (AvaI) IIIb-specific restriction digest; (EcoRV) IIIc-specific restriction digest. (C) RT-PCR reveals that both DT and AT3 cells include IIIc in the pGIIIcI^ΔΔ construct, but only DT cells robustly skip IIIc in the pGIIIcI² construct. Bars indicate the percent inclusion for each sample, and error bars represent standard deviations of the mean. (D) The GIIIcI^ΔΔ and GIIIcI² reporters were stably transfected into DT and AT3 cells. Untransfected cells that did not receive both the reporter and the G418 selection gene were gated away using fluorescence activated cell sorting (FACS). As expected, epithelial-specific IIIc skipping of pGIIIcI² is observed in DT cells, but deletion of IAS2 and ISAR partially abrogates epithelial-specific control of IIIc.