Mass spectrometry
H4 cells were treated with 50 nM calicheamicin or 30 µM camptothecin for 1 h at 37°C and then lysed in nondenaturing lysis buffer (described above). FUS was immunoprecipitated (procedure described above) from the samples using protein G–conjugated Dynabeads (Invitrogen) and the custom produced polyclonal anti-FUS(Ser-30) PAN antibody (Genscript), which recognizes both phosphorylated and unphosphorylated FUS. Samples were enriched for the phosphorylated population of FUS using a titanium dioxide column (ThermoFisher Scientific). The protein was eluted, cleaved with trypsin and then chymotrypsin to isolate the N-terminal prionlike domain of FUS. Mass spectrometry analysis was then performed at the John Hopkins University Mass Spectrometry and Proteomic Facility. This protocol was previously described by Monahan and colleagues (Monahan et al. 2017). PEAKS Studio Software was used to view and analyze the mass spectrometry data.