616 fs* mutation undergoes nonsense-mediated decay. (A) Sanger sequencing results show a single A deletion of the mutated plasmid that produces 616 fs*. (B) Transfection results indicate VCP-eGFP containing 616 fs* expressed at barely detectable levels. (C) Western blot analysis indicates robust expression of wild-type VCP-eGFP fusion protein but undetectable levels of VCP -eGFP with 616 fs* mutation. (D) Quantitative PCR of isolated DNA indicates similar levels of transfected plasmid, implying that reduced expression of VCP-eGFP with 616 fs* is not likely due to differences in transfection efficiency. (E) Sanger sequencing results indicate 616 fs* mutant transcript is partially stabilized by cycloheximide (CHX) plus actinomycin D (AMD) suggesting that it undergoes NMD.