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. 2009 Feb 17;29(8):2155–2167. doi: 10.1128/MCB.01325-08

FIG. 1.

FIG. 1.

Recovery of decay intermediates with trimethyl cap antibody and eIF4E. (A) A total of 40 μg of cytoplasmic RNA from Thal10 cells was incubated with H20 cap antibody bound to protein G-Sepharose. The recovered beads were washed twice with binding buffer (lanes 5 and 6), twice with 50 μM GDP (lanes 7 and 8), and eluted twice with 50 μM m7GDP (lanes 9 and 10). RNA in each eluate was recovered by ethanol precipitation and analyzed by S1 nuclease protection assay using a 5′-labeled antisense probe (top) from nucleotide 250 past the 5′ end of β-globin mRNA. As a control, 8 μg of Thal10 cell cytoplasmic RNA was analyzed in lane 3 (input). A sample of the S1 probe plus 10 μg of yeast tRNA is in lane 1 (probe, −S1), and the same sample after S1 nuclease digestion is in lane 2 (probe, +S1). The positions of HinfI restriction fragments of ΦX174 DNA electrophoresed on the gel (molecular size markers) are indicated on the left, and the identified β-globin mRNA decay intermediates are indicated on the right. (B) Cytoplasmic RNA from Thal10 cells was analyzed as in panel A, with the exception that the RNA was recovered with GST-eIF4E bound to glutathione-Sepharose. (C) The relative amounts of full-length mRNA and each decay intermediate recovered in panels A and B were quantified by a PhosphorImager, and the results were normalized to full-length mRNA and each decay intermediate in the input RNA. (D) Cytoplasmic RNA from Thal10 cells was bound onto immobilized H20 cap antibody, washed as in panel A, and eluted with 50 μM GDP (lane 4) or the indicated concentrations of m7GDP (lanes 5, 6, and 7). (E) Cytoplasmic RNA from Thal10 cells was incubated with immobilized eIF4E in the presence of 0, 0.1, 1, 10, or 100 μM m7GpppG (lanes 5 to 8) or ApppG (lanes 9 to 12). Bound complexes were washed with binding buffer containing 50 μM GDP, eluted with 50 μM m7GDP, and analyzed by S1 nuclease protection assay. The positions of full-length mRNA and each of the decay intermediates are indicated on the right. As a control, 20% of Thal10 cell cytoplasmic RNA was applied to lane 3 (input).