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. 2010 Jan 21;29(4):749–760. doi: 10.1038/emboj.2009.397

Figure 4.

Figure 4

Identification of trans-acting factors that bind the ATM pseudoexon using adipic acid–agarose beads based pull-down and mass-spec analysis. (A) A scheme of the ATM pseudoexon RNAs used for pull-down analysis is presented. The underlined sequence represents the region of the pseudoexon used for pull-down analysis that spans from nucleotide 1–45 for ATM WT and ATMΔ and from nucleotide 32–65 common to both. (B) Identification of proteins that interact with the ATM WT 1–65 and Δ 1–65 pseudoexon RNAs. Different synthetic RNAs were covalently linked to agarose beads and incubated with Hela nuclear extract under splicing conditions. Proteins that remained bound to the RNAs after washing were separated on 10% SDS–PAGE and detected by Coomassie blue staining. The protein bands identified by mass-spec are mentioned with arrowheads on the sides of the gel. To obtain a better quantitative picture, the right panel contains western blots against the U1-70K, U1-A, SF2/ASF, and hnRNP A1 factors (C) Pull-down analysis using RNAs that span from nucleotide 1–45 for ATM WT and ATMΔ and from nucleotide 32–65 common to both. Protein bands identified by mass-spec are mentioned with arrowheads on the sides of the gel.