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. 2007 Nov 5;28(2):601–608. doi: 10.1128/MCB.01341-07

FIG. 1.

FIG. 1.

Recruitment of Aly/REF onto intronless mRNAs. (A) A mixture of 32P-labeled in vitro-transcribed RNAs containing two intronless mRNAs (DHFR and Ftz mRNAs), CTE, U1ΔSm, and U6Δss snRNAs was injected into the nuclei of Xenopus oocytes. The nuclear fraction was prepared after 1 h, and immunoprecipitation (IP) was performed with anti-Aly/REF monoclonal antibody (anti-REF, 11G5; ImmuQuest) (17) or control antibody (anti-Myc, 9E10; Sigma). RNA precipitated with each antibody was recovered and analyzed by denaturing PAGE and autoradiography. The input lanes contained 10% of the input mixture. (B) The same 32P-labeled RNA mixture as for panel A, except that tRNAPhe was supplemented, was incubated in 7.5% HNE, 12 mM HEPES-KOH (pH 7.9), 60 mM KCl, 1.6 mM MgCl2, 0.1 mM EDTA, 6% glycerol at 30°C for 30 min in the absence (−) or presence (+) of 2 mM ATP. After the incubation, immunoprecipitation was performed and the coprecipitated RNA was analyzed as described for panel A.