Rabbit anti fus

Proteins were extracted from head homogenates of approximately 30–50 flies of the respective genotypes. Adult flies were exposed to liquid nitrogen followed by shaking to separate the heads from the bodies. Heads were collected and homogenised in M-PER Mammalian Protein Extraction Buffer (Thermo Scientific) in the presence of protease inhibitor (Roche). Cell debris was removed by centrifugation, and protein concentrations were determined by BCA protein assay (Thermo Scientific). 50–150 µg of total proteins of each samples were denatured by heating to 95 °C and loaded on SDS–PAGE gels. After electrophoresis, proteins were transferred to nitrocellulose paper, blocked with 1 × PBST + 5 % non-fat milk and incubated in primary antibody overnight at 4 °C. This was followed by incubation in secondary antibody. Protein expression was detected by chemiluminescence (Thermo Scientific). Primary antibodies used were rabbit anti-FUS (from Bethyl Labs, cat. #A300-302A, 1:1000 dilution), rabbit anti-N-terminal hFUS (LSC, unpublished result, 1:1000), mouse anti-serotonin (Thermo Scientific, 1:40 dilution), and mouse anti-tubulin (Sigma, 1:10,000 dilution). Secondary antibodies used were HRP-conjugated goat anti-mouse/rabbit (Santa Cruz Biotechnology, Inc., 1:4000 dilution).

Spinal cords of at least 5 animals per genotype were dissected and lysed in RIPA buffer (50 mM Tris HCl pH 7.4, 250 mM NaCl, 1 mM EDTA, 5 mM MgCl2, 1% Triton X-100, 0.25% Na-Deoxycholate, 0.1% SDS, protease inhibitor cocktail). After 2 × 10″ sonication cycles, samples were incubated on ice and then centrifuged at 18000 × g for 20′ at 4 °C. Supernatants were then quantified with Bradford protein assay (Bio-rad) and resuspended in Laemmli Buffer before SDS-PAGE (Sigma-Aldricht). Antibodies used: rabbit anti-FUS (Bethyl), mouse anti-GFAP (Cell Signaling), mouse anti-Iba1 (Wako), rabbit anti-Gemin2 (Proteintech), mouse anti-Sm B/B’ (Thermo Scientific), mouse anti-SMN (BD), mouse anti-β-actin (Sigma).Anti‐rabbit and anti‐mouse IgG peroxidase‐conjugated secondary antibodies were from Bio Rad.

Rabbit anti-FUS (Cat# A300-302A) and anti-Lig1 (A301-136A) antibodies were procured from Bethyl Laboratories, Inc. Mouse anti-FLAG antibody (A8592) was obtained from Sigma-Aldrich, and mouse anti-Lig3 antibody (Cat# ab587) was purchased from Abcam. Mouse anti-Tom20 (SC-17764), anti-HSP60 (SC-13115) and anti-PCNA (SC-56) antibodies were procured from Santa Cruz Biotechnology. Fluorescent secondary antibodies, Alexa Fluor 488 anti-mouse (Cat# A28175), and Texas Red anti-rabbit antibody (Cat# T-2767) were obtained from Life Technologies. The antibodies were diluted at 1:1000 for western blotting, 1:500 for immunofluorescence and 1:100 for PLA.
The human Lig1 coding sequence was re-cloned into pCDNA3.1 plasmid as an N-terminal FLAG and COX8 gene mitochondrial targeting sequence containing construct (FLAG-MTS-Lig1) ^{53 (link)}.