25S rRNA depurination does not activate integrated stress response. (A) Immunoblots of eIF2α (38 kDa) expression and phosphorylation level from WT BY4741 cells transformed with cDNAs of PAP, PAPx, or EV at 0 and at 6 h following induction with 2% galactose. As a positive control for eIF2α phosphorylation, WT BY4741 cells were treated with 30 mM 3-aminotriazole (3-AT) for 90 min. Total cell lysates (60 µg) were separated through 12% SDS PAGE, transferred to nitrocellulose, and probed with total eIF2α (1:1000) and phospho-eIF2α (1:1000) antibodies. The blots shown are representative of three independent biological replicates (n = 3). (B) WT and Δgcn2 BY4741 cells were cotransformed with plasmids encoding cDNAs of PAP, PAPx, or EV, along with p180 and pRS313 and were grown in 2% galactose for 6 h. 3-AT (30 mM) served as a positive control and was added to cells cotransformed with EV, p180, and pRS313 and incubated in 2% galactose for 90 min. β-Galactosidase enzyme activity was quantified using ONPG (O-nitrophenyl β-galactoside) as a substrate. A two-way ANOVA with Tukey's multiple comparisons test was used to determine the statistical significance of differences in β-galactosidase activity. Bars represent means ± SEM of three independent biological replicates (n = 3). The different letters on bars represent statistically significant differences at P < 0.05.
