RSF7, hnRNPA1, and SPF30. A nearly identical set of abundant/moderately-abundant proteins is found in kinetically-stalled A complexes, indicating that A028 is very similar to A complexes formed in the absence of inhibitor. Most U4/U6.U5 tri-snRNPs are intact in the presence of cp028 A block in the conversion of the A complex to the pre-catalytic B complex could arise via disruption of the U4/U5.U6 tri-snRNP. To check if the stability of PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19826927 the U4/U6.U5 tri-snRNP is affected by cp028, we incubated HeLa nuclear extract under splicing conditions in the presence of DMSO or cp028, and then separated the spliceosomal snRNPs by glycerol gradient TG100 115 price centrifugation and determined their distribution by Northern blotting. In the DMSO control, tri-snRNPs peaked in fractions 15 to 18, based on the presence of the U4, U5 and U6 snRNAs. In the presence of cp028, tri-snRNPs sedimented as a broader peak in fractions 15 to 21, suggesting that they may be more prone to aggregation, and only a small increase in the amount of free U5 and U4/U6 snRNPs was observed in the upper fractions of the gradient, indicating that most of the tri-snRNP remains intact. We also performed immunoprecipitations at different salt concentrations with antibodies directed against the U5-116K/ Snu114 protein and determined the extent of coprecipitation of U4 and U6 snRNA. In the presence of cp028 only a small to moderate decrease in the 
amount of U4/ U6 snRNP that coprecipitated with U5 was observed at 190 and 290 mM salt, compared to the DMSO control. Thus, cp028 has only a moderate affect on trisnRNP stability, with most tri-snRNPs still intact under splicing conditions. Compound 028 stalls spliceosome assembly at a stage between B and Bact Affinity-purified complexes formed in the presence of cp028, that were isolated from gradient peak II, contained stoichiometric amounts of U2, U5, U6 snRNA and the MINX-MS2 pre-mRNA, but substantially reduced levels of both U1 and U4 snRNAs, suggesting that they are stalled at the Bact stage after Brr2-mediated dissociation of U4 snRNA. However, human Bact complexes exhibit a slightly higher S-value than B complexes during glycerol gradient centrifugation, and also migrate considerably slower on native agarose gels than B. The B-like complex that accumulates in the presence of cp028 has, in contrast, a lower S-value than B and also migrates faster than a Bact complex on a native gel, suggesting that it is a novel assembly intermediate. MS analysis and 2D gel electrophoresis followed by MS revealed that B028 complexes, like B complexes, contain predominantly U2 and U5 snRNP proteins, as well as most proteins from the group designated B-specific that are recruited at the B complex stage and are no longer abundant/present in the mature human Bact complex. Abundant proteins of purified B028 complexes, as determined by 2D gel electrophoresis, are summarized in Sidarovich et al. eLife 2017;6:e23533. DOI: 10.7554/eLife.23533 5 of 32 Research article Biochemistry Cell Biology Sidarovich et al. eLife 2017;6:e23533. DOI: 10.7554/eLife.23533 6 of 32 Research article Biochemistry Cell Biology recruitment. Proteins from affinity-purified B, B028 or Bact complexes were analysed by Western blotting using antibodies against the indicated proteins. Antibodies against hSnu114 were used to ensure equal loading. Left panel: Western blots with proteins from the B028 peak fractions 1216 of a glycerol gradient on which affinity-purified B028 complexes were fractionated.