Western blotting and immunoprecipitation
Cell lysates were mixed with 4X NuPAGE LDS Sample Buffer (Thermo Fisher NP0008) and run through SDS–PAGE using precast gels (Bio-Rad 456-9034). Protein was transferred onto nitrocellulose membrane (Bio-Rad 162-0146) using the Trans-Blot Turbo Transfer System (Bio-Rad 170-4150). Membranes were blocked in 6% milk (Bio-Rad 1706404XTU) in Tris-buffered saline (TBS) with 0.1% Tween-20 or Odyssey Blocking Buffer (LI-COR 927-40000). Primary and secondary antibodies were diluted in phosphate-buffered saline with 0.1% Tween-20. Secondary antibodies were conjugated to IRDye fluorochromes (LI-COR 926-32211; 925-68020), and their fluorescence was detected using the Odyssey LCx Imaging System (LI-COR). Biological replicates (n) were produced from separately cultured cells. Densitometry analysis was done using the Odyssey LI-COR Image Studio program. All data were displayed as the mean value with error bars representing 95% confidence intervals, unless otherwise stated in the figure legends, using GraphPad Prism version 7.00 for Windows (GraphPad Software, La Jolla, CA, www.graphpad.com).
Protein G–conjugated Dynabeads (Invitrogen 10007D) and 5 µg of antibody were used to immunoprecipitate FUS from mammalian cell lysates following the manufacturer’s protocol. The following FUS antibodies were used: Bethyl A300-293A and custom-produced rabbit anti-FUS(Ser-30) phosphorylated and unphosphorylated peptides (PAN).