Sam68ΔC does not alter the polyadenylation status of the affected viral RNAs. Cells were transfected with pgTat with (+Rev) or without (-Rev) Rev in the absence (pcDNA) or presence of the various Sam68ΔC mutants. Total RNA was harvested and used in the assays below. a) 20 μg of total RNA was selected using oligo(dT) beads and both the polyA- and polyA+ fractions were input into the RNase protection assay. b) RACE-PAT analysis. The illustration at top shows the position of the anchor primer used to make cDNA and the relative positions of the PCR primers. The anchor primer can anneal anywhere along the length of the polyA tail in order to make cDNA, and the resultant PCR generates a smear representing various lengths of polyA tail. Amplicons to either spliced or unspliced RNAs were generated using S F or US F primers, respectively, and the anchor primer. Products were analyzed by fractionation on PAGE gels, and sizes of products determined by comparison to markers.