PAP accumulates in WT BY4741 cells and depurinates 25S rRNA. (A) Time course of PAP expression from WT BY4741 cells transformed with cDNAs of PAP, PAPx, or EV at 0, 3, and 6 h postinduction with 2% galactose. Total cell lysates (60 µg) were separated through 12% SDS PAGE, transferred to nitrocellulose and probed with a PAP-specific antibody (1:10,000). Purified PAP (29 kDa) was used as a standard. RpL3 (43 kDa) was detected using an antibody against RpL3 (1:5000) as a loading control. The blots shown are representative of three independent biological replicates (n = 3). (B) Depurination levels of SRL upon PAP and PAPx expression at 0, 3, and 6 h postinduction with 2% galactose. Total RNA (500 ng) was isolated from cells, and RT-qPCR was performed to measure “reference” and “target” cDNA levels as illustrated in Supplemental Figure S1. Fold changes for PAP and PAPx are shown relative to EV. Differences in depurination levels were tested for statistical significance by two-way ANOVA with Tukey's multiple comparisons test. Bars represent means ± SEM of three independent biological replicates (n = 3). Different letters on bars represent statistically significant differences at P < 0.05.
