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. 2018 Nov 19;115(51):E11904–E11913. doi: 10.1073/pnas.1810413115

Fig. 4.

Fig. 4.

Interaction of NMD factors with UPF1 and RNAs increased in the presence of mutant FUS. (A and B) NMD factors coprecipitated with endogenous UPF1 from N2a cells expressing EV, WT, or mutant FUS. Immunoblots of UPF1, p-UPF1, eRF3b, UPF3b, SMG6, and 3× FLAG-FUS are shown in A, and quantitative results are shown in B. Protein intensities were normalized to corresponding UPF1 bands and compared with EV. (C and D) NMD factors coprecipitated with BrdU-containing RNAs. N2a cells expressing EV, WT, or mutant FUS were incubated with 1 μM BrdU, and RNAs were UV cross-linked to proteins. BrdU IP was performed using an anti-BrdU antibody, followed by Western blots for UPF1, eIF4A3, XRN1, SMG6, FUS, PABP1, and actin. BrU, bromouridine. Quantification of proteins in C is shown in D. Proteins were normalized to the loading control, PABP1, and compared with EV. The purple, green, blue, and red bars represent EV, WT FUS, R495X FUS, and P525L FUS, respectively. Error bars represent SDs for three biological replicates. *P ≤ 0.05; **P ≤ 0.005; ***P ≤ 0.001. Quantifications were compared with EV using a Student’s t test.