Figure 7
Transcriptional activation of Oct-4 downstream target genes by EWS-Oct-4B in vivo. (A) Schematic representation of the expression vectors. Expression vector pCAG-IP/EWS-Oct-4B-EGFP corresponds to EWS-Oct-4B fused to EGFP. The pCAG-IP/EGFP expression vector was used as a control. The CAG expression units (CAG) are indicated by shaded boxes, EWS-Oct-4B is represented by an open box, and EGFP is indicated by solid boxes. (B) Immunoblot analysis of EWS-Oct-4B expression in stably transfected ZHBTc4 ES cells. Total cell lysates (60 μg protein) were fractionated by 12% SDS–PAGE and visualised by western blotting with anti-EGFP (Invitrogen Molecular Probes, top panel) or anti-GAPDH (V-18, Santa Cruz Biotechnology, lower panel) antibodies. (C) Induction of Oct-4 downstream target genes by EWS-Oct-4B in vivo. Northern blot analyses of fgf-4 and nanog mRNAs were performed in ZHBTc4 ES cells expressing vector (lane 1) or EWS-Oct-4B fusion proteins (lane 2). Total RNA was fractionated on a 6% formaldehyde–1.5% agarose gel, transferred to a nylon membrane, and probed using mouse fgf-4 (upper panel) or nanog (second panel) cDNAs, as described in Materials and Methods. Ethidium bromide (EtBr) staining of the agarose gel used for northern blotting shows that equal amounts of total RNA were loaded in each lane (lower panel). The stable cell lines from which the total RNAs used in the northern blot analysis were derived are shown above the panel.
