FIG 2.

Cwc24 stabilizes the association of Prp2 with the spliceosome but is not required for Prp2-mediated spliceosome remodeling. (A) Splicing was carried out in mock-treated (lanes 1 to 4) or Cwc24-depleted (lanes 5 to 12) extracts without (lanes 5 to 8) or with (lanes 9 to 12) prior addition of Cwc24 in the presence of 60 nM V5-prp2-S378L, and the reaction mixtures were precipitated with anti-Ntc20 (lanes 3, 7, and 11) or anti-V5 (lanes 4, 8, and 12) antibody. RXN, 1/10 of the reaction mixture used for immunoprecipitation; PAS, protein A-Sepharose; α-20, anti-Ntc20. (B) Splicing was carried out in Cwc24-depleted Prp9-V5 (middle) or Hsh155-HA (bottom) extracts, and ATP was then depleted. Following addition (lanes 8 to 14) or no addition (lanes 1 to 7) of 10 ng of recombinant Cwc24 and incubation for 5 min, the reaction mixtures were precipitated with anti-Prp2 (top), anti-V5 (middle), or anti-HA (bottom) antibody under standard conditions but using 75 mM instead of 150 mM NaCl in the wash buffer. The precipitated spliceosome was then incubated in splicing buffer with or without ATP for 20 min. Supernatant and pellet fractions were separated and analyzed. R, 1/5 of the reaction mixture used for immunoprecipitation; IP, immunoprecipitation; T, total precipitate; P, pellet; S, supernatant.