Figure 4(a) Prp5-U2 co-immunoprecipitation (IP). Wild-type (lanes 1 and 2) and snR81Δ pus1Δ (lane 3) strains, each containing a FLAG-tagged PRP5 gene (indicated on the top), were used for anti-FLAG IP in the absence (lane 1) or presence (lanes 2 and 3) of ATP. The precipitated Prp5 (WT Prp5-FLAG) as well as co-precipitated U1 and U2, detected by primer-extension, are indicated.(b) Quantification of co-precipitated U2 snRNA. For each lane [the WT lane and the snr81Δ pus1Δ lane, corresponding to lane 2 and lane 3 of (a), respectively], Prp5-co-precipitated U2 was normalized against co-precipitated U1 [(intensity of the U2 band) / (intensity of the U1 band)]. The snr81Δ pus1Δ lane was then further normalized against the WT lane [(the value of normalized U2 from the snr81Δ pus1Δ lane) / (the value of normalized U2 from the WT lane)]. Quantification was based on three independent experiments.(c) U2 expression level in wild-type and snR81Δ pus1Δ strains. Total RNA, isolated from the wild-type (lane 1) or snR81Δ pus1Δ (lane 2) strains, was used for primer-extension to measure the levels of U2 and U6. The U2 and U6 bands are indicated.d) Depletion of Prp5. Wild-type (lanes 1, 3 and 4) and snR81Δ pus1Δ (lanes 2 and 5) cells containing a FLAG-tagged PRP5 were lysed, and anti-FLAG IP was performed at a high concentration of salt. Cell extracts, before (lanes 1 and 2), and after [lanes 3 (mock), 4 and 5] anti-FLAG IP, were used for western analysis using anti-FLAG. The Prp5 band is indicated.(e) IP of reconstituted Prp5 (GAR)-U2. An equal amount of FLAG-tagged Prp5 (GAR) was added to the wild-type (lane 1) and snR81Δ pus1Δ (lane 2) cell extracts depleted of endogenous Prp5 [see (c)]. After the salt concentration was brought back to its original level, anti-FLAG IP was carried out. The Prp5 (GAR) band, detected by western, is indicated. The co-precipitated U1 and U2 bands, detected by primer-extension, are also indicated.f) Quantification of U2 that is co-precipitated with mutant Prp5 (GAR). For each lane [including the WT lane and the snr81Δ pus1Δ lane, corresponding to lane 1 and lane 2 of (e), respectively], U2 co-precipitated with Prp5 (GAR) was normalized against co-precipitated U1 [(intensity of the U2 band) / (intensity of the U1 band)]. The snr81Δ pus1Δ lane was then further normalized against the WT lane [(the value of normalized U2 from the snr81Δ pus1Δ lane) / (the value of normalized U2 from the WT lane)]. Quantification was based on three independent experiments.(g) Prp5-U2-pre-mRNA co-IP. Reconstitution was carried out as described in (d). Prior to IP, an equal amount of radiolabeled pre-mRNA was also added to each reaction. Anti-FLAG IP was then performed in the presence of ATP. The precipitated Prp5 (WT and GAR) band, detected by western, is indicated. The co-precipitated pre-mRNA, directly visualized after electrophoresis, is also indicated. The pre-mRNA input is also shown. In lanes 1 and 2, wild-type and snR81Δ pus1Δ cell extracts, depleted of Prp5, were reconstituted with the wild-type FLAG-Prp5. In lanes 3 and 4, wild-type and snR81Δ pus1Δ cell extracts, depleted of Prp5, were reconstituted with mutant FLAG-Prp5 (GAR).(h) Quantification of pre-mRNA co-precipitated with Prp5 and U2. For each lane in (g), pre-mRNA co-precipitated with Prp5 (Wild-type or GAR) was normalized against the Prp5 (WT/GAR) band in the same lane (detected by Anti-FLAG) [(intensity of pre-mRNA) / (intensity of WT/GAR Prp5)]. Lanes 2,3,and 4 (see (g)) were then further normalized against lane 1 (see (g)) [(the value of normalized pre-mRNA from lane 2, 3 or 4 in (g)) / (the value of normalized pre-mRNA from lane 1 in (g))]. The numbers at the bottom of (h) correspond to the lane numbers of (g). Quantification was based on three independent experiments.