Figure 5.

Effects of the mutations of ALAS2int1GATA on GATA1-binding activity. (A) DNA probes used in the EMSA. The nucleotide sequences in the antisense strand of the probes are shown. The position of each probe is also indicated in Figure 1B as the solid horizontal bar. ALAS2int1GATA is boxed in the sequence of the wt probe, and the single nucleotide transition (GGTA mutation) is underlined in the sequence of the GGTA probe. The delGATA probe represents the 5′- and 3′-flanking sequences of the deleted 35-bp segment (see Figure 3B). (B) Effect of each mutation of ALAS2int1GATA on GATA1-binding activity. Wild-type probe (lanes 3–7) or each mutant probe (lanes 8, 9) was incubated with the nuclear extracts prepared from HEK293 cells transfected with the GATA1-FLAG expression vector. An excess amount of unlabeled wild-type probe (lane 5), each of the unlabeled mutant probes (lanes 6, 7), or anti-FLAG antibody (lane 4) was included in the reaction mixture. Lane 2 shows the negative control with nuclear extracts from HEK293 cells transfected with mock vector.