Fig. 6. Smad5 knockdown in mouse EBs results in reduced Eklf and Gata1 expression. (A) Schematic of the GFP-Intron-miR transgene after site-specific, uni-directional integration into the Ainv18 homing site, including the location of the miR-30 backbone and shRNA insertions into the GFP intron. (B) Flow cytometric analysis of GFP expression in the Smad5 shRNA-1 Ainv18 ES cell clone in doxycycline treated (24 hours, +dox) or untreated (-dox) EBs harvested at day 5 of differentiation. (C) Analysis of Smad5, Eklf and Gata1 expression by qRT-PCR in the Smad5 shRNA-1 clone. Untreated (-dox) or doxycycline-treated (24 hours; +dox) cells were harvested at day 5 of EB differentiation. To enrich for transgene-expressing populations, cells from untreated EBs were FACS sorted for GFP-negative cells (-dox/GFP^-), whereas cells from dox-treated EBs were FACS sorted for GFP expression (+dox/GFP⁺) prior to being monitored for RNA expression. Expression levels were normalized to Gapdh and untreated samples were set to 1. (D) Analysis of Smad1, Smad5, Eklf, Gata1 and Gata2 expression by qRT-PCR in the shRNA-1 clone in cells harvested from EBs at day 5 of differentiation without FACS sorting. RNA was isolated from untreated (-dox) or doxycycline-treated (24 hours, +dox) cells as indicated. Expression levels were normalized to Gapdh and each untreated sample was set to 1. Arithmetic mean ±s.d. (E) Western blot analysis of Gata2, Eklf, Gata1 and GFP protein expression in shRNA-1, shRNA-2 and shRNA-control Ainv18 ES cell clones harvested at day 6 of EB differentiation. Lysates were prepared from unsorted EBs that had been treated with (+dox) or without (-dox) doxycycline for 48 hours. Hsp90 expression levels were used as a loading control. Molecular mass (kDa) markers for each blot are shown to the left.