To gain further insight into the role of SF2/ASF in controlling alternative splicing of CD200, an siRNA against SF2/ASF was employed to knock down SF2/ ASF in Daudi and SK-N cells. A scramble siRNA was used as a negative control. After Figure 3 . The pattern of expression of exogenous full-length CD200 or truncated CD200 in different cells parallels that of the endogenous molecules and mutation of the ESE in exon 2 abolishes exon 2 inclusion. (A) The location of the primers used for real-time RT-PCR. Primers 1 and 2 were used for endogenous expression of full-length CD200; primers 3 and 4 were used for endogenous expression of truncated CD200; primers 5 and 6 were used for exogenous expression of full-length CD200; primers 7 and 8 were used for exogenous expression of truncated CD200; primers 9 and 10 were used for the constitutive expression of V region of CD200. (B) The specificity of the primers used for full-length or truncated CD200. CD200 RNA containing exon 2 or lacking exon 2 from in vitro transcription was reverse transcribed and used for real-time PCR using the primer pairs labeled in the figure. (C) Mutation of the ESE in exon 2 was confirmed by DNA sequencing. (D) Endogenous and exogenous expression of the full-length and truncated CD200 in Daudi cells, and exogenous expression of two isoforms after mutation of the ESE. (E) Endogenous and exogenous expression of the full-length and truncated CD200 in SK-N cells, and exogenous expression of two isoforms after mutation of the ESE. The data represent the mean ± SE (three independent experiments, triplicate determinations). Broken lines reflect exogenous expression of the full-length CD200 decreased after mutation of the ESE relative to that of wild type (P < 0.01 in Daudi; P < 0.05 in SK-N). Continuous lines reflect exogenous expression of the truncated CD200 increased after mutation of the ESE relative to that of wild type in SK-N cells (P < 0.05). and total RNAs extracted for real-time RT-PCR, along with nuclear proteins for western blot. As shown in Figure 6A , SF2/ASF expression was eliminated after treatment with 2.5 mg of siRNA. b-Actin was used as a loading control. Real-time RT-PCR was then performed using RNA samples treated with siRNA. As shown in Figure 6B and C, the endogenous expression of full-length CD200 (exon 2 inclusion) was reduced in both Daudi and SK-N cells, compared with Mock (no siRNA) or scramble siRNA-treated cells. The same pattern was observed for the exogenous expression of CD200 in Daudi ( Figure 6D or SK-N cells ( Figure 6E ) following silencing SF2/ASF. Consistent with the observation resulting from the ESE mutation or deletion, the expression pattern of full-length versus truncated CD200 was reversed in SK-N cells after knockdown of SF2/ASF.