FIG 3.

Cwc24 has an essential function prior to Prp2 action. (A) Western blotting of wild-type (lane 1), metabolically Cwc24-depleted (lanes 2 and 3), and Spp2-depleted (lane 3) extracts. (B) Splicing was carried out in mock-treated (lanes 1 to 4) or Spp2-depleted (lanes 5 to 13) metabolically Cwc24-depleted extracts without (lanes 3, 4, and 9 to 13) or with (lanes 2 and 6 to 8) the addition of Cwc24 and/or Spp2 for 30 min. To the extracts without prior addition of Cwc24 or Spp2, either Cwc24 (lanes 12 and 13) or Spp2 (lanes 10 and 11) was then added and incubated for 5 min, followed by addition of the other reciprocal protein and incubation for 5 min (lanes 10 and 12) or 10 min (lanes 11 and 13). For the mock-treated reaction mixture, Cwc24 was added and incubated for 5 min (lane 4). Δ24, Cwc24 metabolically depleted extracts; d2, Spp2 depletion; Sp, splicing; +24, Cwc24 added; +2, Spp2 added.