Biochemical Analysis of Protein Binding to UAGG Silencer Motifs as a Function of Cell Excitation

<div><p>(A) Schematic of methodology used to analyze protein binding to RNA substrates in nuclear extracts from KCl-induced and mock-treated cultures. UV crosslinking assays were performed under splicing conditions to monitor protein binding to radiolabeled RNA oligos (lanes 1–8), and to the full-length CI cassette exon (lanes 9 and 10). Sequences of RNA oligos and schematic of CI exon are shown at right of gel panels. Gel panels show radiolabeled proteins after SDS-PAGE separation; kDa ladder indicates molecular weight standards.</p> <p>(B) Affinity selection of proteins non-covalently bound to RNA substrates containing the full-length CI cassette exon with (M3_E18) or without (E18) the M3 hairpin. Assembly reactions containing RNA substrates pre-bound to MS2-MBP were incubated under splicing conditions, bound to amylose columns, and eluted with maltose. Samples were resolved by SDS-PAGE and transferred to nylon membranes for immunoblotting with hnRNP A1-specific 9H10 (lanes 11–16) or ASF/SF2 (lanes 17–22) antibody. A1 and A1^B represent the major and minor isoforms of hnRNP A1, respectively.</p> <p>(C) Quantitative Western blots show serial dilutions of recombinant hnRNP A1 (MBP-A1) grown in Escherichia coli as reference standards from 1 to 20 ng (top panel). Nuclear levels of hnRNP A1 and A1B are shown at three loading levels, 100, 200, and 400 ng of total nuclear extract (NE) protein (bottom panel). Nuclear extracts were prepared from mock-treated (Mock) and 50 mM KCl-treated cultures (Depol). Monoclonal antibody 9H10 was used to detect hnRNP A1 in both blots.</p>