SRm160/300 is required for ESE-dependent splicing. The splicing activity of a Drosophila doublesex (dsx) pre-mRNA that is dependent on a GAA-repeat ESE in the 3′ exon was assayed in the presence or absence of SRm160/300. Splicing reaction mixtures containing SRm160/300-immunodepleted nuclear extract (lanes 1, 3, and 5), or nuclear extract mock-depleted with preimmune serum (lanes 2, 4, and 6), were incubated for 1 hr with a dsx pre-mRNA with no enhancer sequence (dsxΔE; lanes 1 and 2), with a dsx pre-mRNA containing three GAA repeats [dsx(GAA)3; lanes 3 and 4], or with a dsx pre-mRNA containing six GAA repeats [dsx(GAA)6; lanes 5 and 6]. The splicing reaction intermediates and products are indicated on the right of the gel.