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. 1999 May 25;96(11):6125–6130. doi: 10.1073/pnas.96.11.6125

Figure 2.

Figure 2

ESE-dependent association of SRm160/300 with the dsx pre-mRNA. (A) Association of SRm160/300 with GAA repeats. Equal amounts of the dsxΔE pre-mRNA and a (GAA)12 or (GUU)12 RNA (lanes 2 and 5) were incubated together in nuclear extract under splicing conditions for 40 min. Complexes assembled on these RNAs were immunoprecipitated with mAb-B1C8 or rAb-SRm300. The input RNAs are shown in lanes 1–3. RNA recovered directly after incubation in nuclear extract is shown in lanes 4–6, and RNA recovered after immunoprecipitation is shown in lanes 7–11. Immunoprecipitations were performed with a nonspecific control Ab (rabbit anti-mouse, lane 7; the corresponding input and total are in lanes 3 and 6, respectively), mAb-B1C8 (lanes 8 and 9), or rAb-SRm300 (lanes 10 and 11). The corresponding inputs for the latter two sets are in lanes 1 and 2, and the corresponding totals are in lanes 4 and 5. The RNA samples were separated on a denaturing 15% polyacrylamide gel. Loading was 12.5% of the total amount of RNA from the inputs and totals and 50% of the total amount of RNA recovered from the pellets (Pels). (B) The association of SRm160/300 with the dsx pre-mRNA requires U1 snRNP in addition to ESE-bound factors. Immunoprecipitations were performed with mAb-B1C8 (lanes 6–8, 15–17, 24–26) or rAb-SRm300 (lanes 9–11, 18–20, 27–29) from sets of splicing reaction mixtures incubated for 40 min containing either a control (mock-depleted) nuclear extract (lanes 1–11), a U1 snRNP-depleted nuclear extract (lanes 12–20), or a U2 snRNP-depleted nuclear extract (lanes 21–29). Each set of reactions was incubated with the three dsx pre-mRNAs described in Fig. 1, as indicated above the gel. RNA recovered directly from splicing reactions (lanes 1–4, 12–14, 21–23) and RNA recovered after immunoprecipitation (lanes 5–11, 15–20, 24–29) was separated on a denaturing 7% polyacrylamide gel. The amounts of RNA loaded are as described for A. A control immunoprecipitation was performed with preimmune serum (lane 5) from a reaction mixture containing mock-depleted nuclear extract and the dsx(GAA)6 pre-mRNA. The corresponding total is shown in lane 4.