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. 2010 Jul 15;24(14):1491–1495. doi: 10.1101/gad.1930710

Figure 3.

Figure 3.

Increased Alr1p expression accounts for the reduced termination fidelity of NMD-deficient cells. (A) Western blot analysis of the 3HA-ALR1 alr2Δ (MJY455), 3HA-ALR1 alr2Δ upf1Δ (MJY456), PMET3-3HA-ALR1 alr2Δ (MJY430), and PMET3-3HA-ALR1 alr2Δ upf1Δ (MJY432) strains. The strains were grown in SC-cys medium containing 300 μM met, harvested and washed with water, diluted to OD600 ∼0.1 in fresh SC-cys (300 μM met) and SC-met-cys medium, and allowed to grow for two generations. Monoclonal antibodies against HA or Pgk1p were used to detect the indicated proteins. (B) Mg2+ levels in selected strains. Cells were grown as described above and assessed for Mg2+ levels by using eriochrome blue SE, and the resulting values were expressed relative to that obtained for the 3HA-ALR1 alr2Δ strain (300 μM met), which was set to 1. Strains are numbered as in A. The values represent the average of 10 independent experiments. The standard deviation is indicated. An asterisk indicates that the value is significantly different from 1 using the Wilcoxon signed rank test (P < 0.05). (C) Readthrough of the UAA codon in strains shifted to medium with or without 300 μM met. Cells were manipulated as in A, except that the medium also lacked uracil to allow selection for the reporter plasmids. Strains are numbered as in A. The values represent the average of five independent experiments. The standard deviation is indicated.