Detection of the peptide derived from an NMD-sensitive XUT reporter. (A) Schematic representation of the tagged xut0741-b alleles, using the same color code as in Figure 5A. (B) WT and upf1Δ cells expressing the native XUT0741 or the xut0741-b allele fused to a C-terminal 3FLAG tag (xut0741-b-FLAG) were grown to mid-log phase, at 30°C, in YPD medium. Total RNA was extracted and analyzed by northern blot. XUT0741 and scR1 were detected as described in Figure 1A. (C) WT and upf1Δ cells expressing the native XUT0741, the xut0741-b-FLAG or the SL-xut0741-b-FLAG alleles were grown as above. Protein extracts (40 μg) were separated by poly-acrylamide gel electrophoresis and then transferred to a nitrocellulose membrane. The size of the protein ladder bands is indicated on the left of the panel. Pgk1 was used as a loading control. (D) WT, upf1Δ and xrn1Δ cells expressing the SL-xut0741-b-FLAG allele were grown as above. For the WT strain, a sample of cells was also treated for 15 min with CHX (100 μg/mL, final concentration). After total RNA extraction, the levels of the SL-xut0741-b-FLAG transcript were assessed by strand-specific RT-qPCR, normalized on scR1 and set as 1 for the untreated WT condition (indicated by the dashed line). Mean and SD values were calculated from three independent biological replicates. (**) P < 0.01; (*) P < 0.05; (ns) not significant upon t-test.
