Ab154141

Lysates were mixed with 4× NuPAGE LDS Sample Buffer (Thermo NP0008) and run through AnyKD precast gels (BioRad 4569034) at 80 V for 2 h. Gels were transferred through either a Trans-Blot Turbo Transfer System (Bio-Rad 1704150) or eBlot L1 (GenScript L00686) onto nitrocellulose membranes (BioRad 1620112). Membranes were blocked with 6% milk (BioRad 1706404) in Tris-buffered saline (TBS). Primary and secondary antibodies were diluted in TBS with 0.1% Tween-20 (Sigma P7949). The following primary antibodies were used to probe the blots: FUS antibodies (Santa Cruz 373698, Abcam ab154141, Bethyl A300-293A, custom rabbit phospho-FUS antibodies), gamma tubulin (Sigma T6557), and GFP (Roche 11814460001). Primary antibodies were detected with secondaries conjugated to IRDye fluorescent probes (LI-COR 926-68021, 926-32210). Blots were imaged with the Odyssey CLx imaging system (LI-COR). Band densitometry quantification was done using Image Studio software (Li-COR). Phosphobands were normalized to endogenous FUS band intensity.

Worms were lysed in protein lysis buffer [50 mM tris-HCl (pH 7.8), 150 mM NaCl, 1% sodium deoxycholate, 1 mM EDTA, 0.1% SDS, 1% Triton X-100, and protease inhibitor (Roche)] using a Precellys 24 homogenizer. Worm lysates were centrifuged at 10,000 rpm for 10 min at 4°C, and the supernatant was collected. Protein concentrations were determined with standard BCA (bicinchoninic acid assay) protein assay (Thermo Fisher Scientific). Twenty micrograms of total protein was separated by SDS–polyacrylamide gel electrophoresis (PAGE), transferred to nitrocellulose membranes (Millipore), and subjected to immunoblotting. Western blot analysis was performed with anti-GFP antibody (1:5000; AMSBIO, TP401), FUS (1:1000; Abcam, ab154141), TDP43 (1:1000; Abcam, ab225710), and α-tubulin (1:20,000; Sigma-Aldrich, T6199).

C. elegans strains were grown from hatching on HT115 E. coli carrying either empty vector or RNAi clones. At day 3 of adulthood, worms were collected with M9 buffer, and worm pellets were frozen with liquid N2. Frozen worm pellets were thawed on ice, and worm extracts were generated by glass bead disruption on ice in nondenaturing lysis buffer [50 mM Hepes (pH 7.4), 1 mM EDTA, 150 mM NaCl, and 1% Triton X-100] supplemented with EDTA-free protease inhibitor cocktail (Roche). Worm debris was removed with 8000g spin for 5 min. One hundred micrograms of protein extract was supplemented with SDS at a final concentration of 0.5% and loaded onto a cellulose acetate membrane assembled in a slot blot apparatus (Bio-Rad). Then, the membrane was washed with 0.2% SDS and SDS-resistant protein aggregates were assessed by immunoblotting using antibodies against GFP (1:5000; AMSBIO, TP401), FUS (1:1000; Abcam, ab154141), and TDP43 (1:1000; Abcam, ab225710). Extracts were also analyzed by SDS-PAGE/Western blot with anti-GFP, anti-FUS, anti-TDP43, and anti–α-tubulin (1:20,000; Sigma-Aldrich, T6199).