Figure 2. DUX4 destabilizes UPF1 via the proteasome.
(A) Relative mRNA levels of NMD factors in DUX4-expressing vs control myoblasts (54-1 cells). Red, up-regulation by ≥1.5-fold. (B) Immunoblot for NMD factors UPF1, SMG7, and UPF3B in DUX4-expressing and control myoblasts (54-1 cells) at 36 hr post-infection. H3, histone H3 (loading control). (C) Immunoblot of total protein from a 36-hr time course of DUX4-expressing and control myoblasts (54-1 cells). H3, histone H3. Loading Control, a nonspecific band that serves as an additional loading control. (D) Quantification of UPF1 protein level from the immunoblot presented in (C), normalized to the nonspecific band that serves as a loading control. (E) Relative levels of transcripts produced from the NMD(+) and NMD(−) β-globin reporter plasmids. (F) Isoform ratios of endogenously produced NMD-degraded isoforms of SRSF2 and SRSF3. Time course identical to (C). Error bars, standard deviation. (G) Immunoblot of total protein from DUX4-expressing and control myoblasts (54-1 cells) treated with the proteasome inhibitor MG132 (10 µM; 8 hr treatment initiated 16 hr after infection with lentiviral expression constructs). Loading control H3, histone 3, has a long half-life (Toyama et al., 2013). (H) Quantification of UPF1 protein levels from three independent replicates of the immunoblot presented in (G), normalized to the nonspecific band that serves as a loading control. Error bars, standard deviation.