A3H deaminase activity dependence on RNase A treatment of extracts and substrate specificity. (A) Western blot analysis showing the WT A3H protein levels in 293T cells. Transfection of the empty vector (no A3H) served as a negative control and showed that 293T cells do not contain detectable levels of endogenous A3H. The tubulin loading control is also shown. (B) Representative gel illustrating assay of WT A3H deaminase activity in a cell extract using a 40-nt TTCA-containing oligonucleotide substrate. The oligonucleotide was incubated with increasing amounts of A3H extract in the presence and absence of RNase A. The positions of the substrate (40 nt) and the deamination product are indicated by arrows to the right of the gel. Lane 1, empty vector control; lanes 2 and 6, lanes 3 and 7, lanes 4 and 8, and lanes 5 and 9 represent reactions containing 1 μg, 2 μg, 3 μg, and 5 μg of total protein, respectively. (C) The percent (%) deamination product was calculated as described in Methods and was plotted against the amounts of total protein. (D) Deaminase assay using WT A3H extract and 40-nt oligonucelotides containing the following deaminase motifs: TTCA, TTCT, TTCG, TGCA, and ACCCA. The data were analyzed and plotted as described in (C).