Analysis of E4BP4 in VWF-producing cells. (A) EMSA and supershifts with the E4BP4 antibody (Ab) using 10 μg of nuclear proteins isolated from BAECs, and undifferentiated and differentiated Dami cells. Oligonucleotides probes contain the E4BP4 consensus element (E4BP4) and the VWF E4BP4 element (Neg 5). NS indicates the presence of a nonspecific DNA/protein complex, and the free probe and well origin are shown. (B) Western blot analysis of nuclear proteins (20 μg/lane) using the E4BP4 antibody (left panel). The estimated mass in kilodaltons of the different forms of E4BP4 is indicated on the left side of the panel. The right panel shows nonspecific bands present when the E4BP4 antibody is preincubated with the blocking peptide. Lane 1 shows HepG2 transfected with E4BP4; lane 2, 10 μg BAECs transfected with E4BP4; lane 3, HepG2; lane 4, BAECs; lane 5, HUVECs; lane 6, undifferentiated Dami; and lane 7, differentiated Dami. (C) Reverse-transcribed and PCR-amplified RNA isolated from the following: lane 1, BAECs; lane 2, HUVECs; lane 3, differentiated Dami; lane 4, undifferentiated Dami; lane 5, HUVECs; lane 6, BAECs; and lane 7, HepG2. A DNA size marker (GeneRuler 100 bp DNA Ladder Plus MBI) is in lane 8. Below is a diagrammatic representation of the E4BP4 RNA with the single intron and the translational start site indicated. The amplified fragments and sizes are also shown. Lanes 1 and 2 were amplified using primers E4BP4-nt-562-Sense and E4BP4-nt-1521-Antisense. Lanes 3 to 7 were amplified with E4BP4-nt-2-Sense and E4BP4-nt-1521-Antisense.