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. 2001 Jul 15;29(14):3087–3098. doi: 10.1093/nar/29.14.3087

Figure 1.

Figure 1

Analysis of C/EBPβ mRNA. (A) Schematic presentation of the C/EBPβ wild-type (pT7-βmtAUG3) constructs used to produce mRNAs by in vitro transcription (B). (B) C/EBPβ wild-type and mutant mRNAs were prepared using the constructs shown in (A). In vitro translation of capped and uncapped wild-type (lanes 1) and mutant (lanes 2) mRNAs are shown. The reaction (25 µl) was started by addition of 1 µg mRNA in the presence of [35S]methionine, and the translation products were resolved by 12% SDS–PAGE. The gels were dried and autoradiographed. (C) C/EBPβ mRNA was analyzed by affinity selection of capped mRNA via the 5′-cap structure. The affinity matrix was generated by binding GST/elF-4E fusion protein to gluthathione agarose. Specific elution of bound mRNA was achieved using saturating amounts of the cap analog m7GTP. The results are shown for non-capped and capped in vitro transcribed mRNA and poly-(A) RNA from COS-1 cells transfected with pCMV-C/EBPβ. Column fractions were collected, concentrated, and subjected to agarose gel electrophoresis and northern blot analysis using a C/EBPβ-specific hybridization probe. Lane, 1, flow through; lanes 2–5, 1× BB washers; lane 6, 1× BB containing 50 µM GTP; lanes 7 and 8, 1× BB containing 50 µM m7GTP; lane 9, HCB wash.