Figure 5.

FBP21 is found in an A/B complex of spliceosome-specific proteins. (A) FBP21 is associated with the A/B complex. RNA binding proteins were assembled on AdML pre-mRNA. RNA-protein complexes were size-selected and purified. Two complexes were isolated: the A/B complex [containing spliceosome-specific proteins (snRNPs)] and the H complex (hnRNPs). Nuclear extract and the two complexes were run on an SDS/PAGE gel, were blotted onto nitrocellulose, and were incubated with α21Ab (Upper). The A/B complex isolation was repeated by using biotinylated [Bio (+) A/B] RNA and nonbiotinylated RNA [Bio (−) A/B]. Samples were run on a SDS/PAGE gel, were blotted onto nitrocellulose, and were incubated with α21Ab (Lower). In both upper and lower, the protein band migrates as 58 kDa. (B–D) 2D gel analyses of spliceosome-specific proteins. Spliceosome-specific proteins were fractionated by 2D gel electrophoresis and were blotted to nitrocellulose. (B) The filter first was probed with α21Ab. (C) It then was reprobed with an antibody against SAP 61, without removing the α21Ab. All blots were developed by using enhanced chemiluminescence (ECL, Amersham). (D) The same filter then was gold stained. FBP21 is indicated by the closed arrowhead, and SAP 61 is indicated by the open arrowhead. Marker (M) and HeLa cell nuclear extract (NE) were run in the second dimension.