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. 2013 Jun 27;8(6):e68291. doi: 10.1371/journal.pone.0068291

Figure 1. MARVELD1 is required for the stabilization of the NMD reporter transcript.

Figure 1

(A) Expression analysis of MARVELD1 by western blotting. H520 cells were transiently transfected with two independent siRNAs against MARVELD1 or a negative control siRNA. A549 and HEK293 cells were stably transfected with pcDNA3.1-MARVELD1-V5/His (HEK293/MARVELD1) or the control plasmid pcDNA3.1-V5/His (HEK293/Vector). (B) A549, HEK293, HeLa and H520 cells as well as MARVELD1-depleted H520 cells, A549/MARVELD1 cells and HEK293/MARVELD1 cells were transiently cotransfected with either pmCMV-Gl Norm or pmCMV-Gl Ter and phCMV-MUP. The mRNA level of β-globin was assessed by real-time PCR of cDNA. The β-globin mRNA was normalized to the expression of Mup mRNA, and the normalized level of G1 (β-globin) Norm mRNA was defined as 1.0. (C) HeLa cells with depleted MARVELD1, overexpressed MARVELD1 or transiently depleted UPF1 were cotransfected with either pmCMV-Gl Norm or pmCMV-Gl Ter and phCMV-MUP, and the level of β-globin mRNA was assessed by treating cells with DRB and serially assessing globin mRNA levels by real-time PCR of cDNA. The β-globin mRNA was normalized to the expression of Mup mRNA, and the normalized level of G1 (β-globin) Norm and Ter mRNA at 0 h was defined as 1.0. The effects of siRNA-mediated knockdown of UPF1 and MARVELD1 were determined by western blotting, and GAPDH was used as an internal control. All of the experiments were performed with three replicates. The average ± S.E. (error bars) is displayed in column diagrams of (B) and (C).