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.