Las 4000 mini

For western blot analysis, cells were washed with cold PBS and lysed in RIPA buffer (Thermo Fisher Scientific, Grand Island, NY, USA). Proteins were quantified using the Bradford method and equal amounts of protein were resolved by SDS-PAGE and analyzed by western blotting as previously described^{33 (link)}. The membranes were incubated with primary antibodies against cleaved PARP, caspase3, survivin (Cell Signaling Technology, Beverly, MA, USA) and β-actin (Sigma-Aldrich, St. Louis, MO, USA) overnight at 4 °C and with a secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h at room temperature. Proteins were visualized using enhanced chemiluminescence (Thermo Fisher Scientific). Western blot images were analyzed with a LAS-4000 mini (GE Healthcare Life Sciences, Uppsala, Sweden) and Bio-Rad ChemiDoc (Bio-Rad, Richmond, CA, USA).

Epstein Barr Virus (EBV) transformed cell lines were produced from three healthy donors (controls) and from three individuals who are homozygous for the TLE6 S510Y mutation (patients, see below). Western blot analysis was performed as described [17 (link)]. Briefly, cells were harvested by centrifugation and resuspended in lysis buffer (20 mM Tris pH 7.5, 350 mM NaCl, 0.05 % β-mercaptoethanol) supplemented with a protease inhibitor cocktail. After sonication and centrifugation, 30 μg of total cell lysates were analyzed by SDS-PAGE on 10 % acrylamide or on 8 % Phospho-tag acrylamide gels (Wako, TX, USA), followed by transfer of the proteins onto nitrocellulose membrane. After blocking in 5 % milk in TBS-Tween, the membranes were incubated with anti-TLE6, anti-KDHC3L/Ecat1 from (Abcam, Cambridge, MA, USA) or anti-OOEP, anti-Flag and anti-GAPDH from (Santa Cruz, CA, USA). After washing, secondary reactions were carried out with biotin conjugated secondary antibodies followed by anti-avidin-HRP conjugated antibody. Signals were visualized using an LAS 4000 mini (GE Healthcare, UK) and quantified using ImageQuant software (GE Healthcare, UK).

HEK293 cells, SH-SY5Y cells, or 10 heads of 1-day-old male flies were lysed in a Laemmli sample buffer with PhosSTOP phosphatase inhibitor cocktail (Roche). The lysates were separated by 7.5 or 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes. After probing with primary antibodies, the immunoblots were developed using a chemiluminescence kit (Wako) or SuperSignal West Femto Chemiluminescent Substrate (Thermo Fisher Scientific), and visualized by LAS-4000 mini (GE Healthcare). The band intensities of FUS protein derived from HEK293 cells were quantified by ImageQuant (GE Healthcare). The band intensities of FUS or α-tubulin protein derived from the heads of each tg fly line were quantified by ImageQuant, and the average relative level of FUS (FUS/α-tubulin) was calculated in three or five independent experiments.

The cell lysates in IP buffer (150 mM NaCl, 1% Nonidet P-40, 1 mM EDTA, 50 mM HEPES, pH 7.4 and protease inhibitor cocktail) reacted with monoclonal antibodies overnight at 4 °C on a rotator and reacted to 50% slurry of protein A/G agarose bead (Pierce) for 2 h at room temperature on a rotator. The protein A/G beads capturing antibody-antigen complex were washed 3 times with PBS then mixed with SDS sample buffer and incubated for 5 min at 99 °C. The eluted supernatant was analyzed by western blot using PVDF membrane (Pall, FluoroTrans). After the membranes were incubated with 5% skim milk in 0.1% TritonX-100 in PBS (PBS-T), and reacted to polyclonal rabbit antibody IM1014 (Calbiochem) for human protein or polyclonal rabbit anti-epithin serum [35 (link)] for mouse protein. HRP-conjugated anti-rabbit IgG (1:5000 diluted) was used with Luminol/Enhancer solution and Stable Peroxide solution (SuperSignal^R West Pico, Thermo). The chemiluminescence was observed using LAS-4000mini (GE healthcare Life Sciences) and signals of each band were digitized by ImageJ.

Proteins were separated by SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked for 1 hr at room temperature with 5% milk in PBS containing 0.05% Tween-20 (PBS-T) and immunoblotted with primary antibodies diluted in PBS-T + 3% milk for 1 hr at room temperature or 16 hr at 4°C. After washing with PBS-T, the membranes were incubated with an appropriate HRP-conjugated secondary antibody. Immunoreactive bands were detected with ECL Select Western Blotting Detection Reagents (GE Healthcare) or Immobilon Western Detection Reagents (Millipore) and captured using a luminescent image analyzer (LAS-4000 mini, GE healthcare).

CTOSs were lysed with RIPA buffer (10 mM Tris-HCl pH 7.4, 1 mM EDTA, 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS) containing EDTA-free complete proteinase inhibitor (Roche) and PhosSTOP phosphatase inhibitor (Roche) at 4 °C. After sonication and centrifugation, the supernatant was collected. The protein concentration was measured using the BCA protein assay kit (Pierce, Waltham, MA). Basically, 10 μg of protein for detection of phosphorylation, 20 μg of protein was applied to one lane. After electrophoresis and blotting, the blots were blocked with 5% BSA in TBS-T (20 mM Tris-HCl pH 7.6, 150 mM NaCl, 0.1% Tween-20) at room temperature, and then incubated with primary antibody in TBS-T containing 1% BSA overnight at 4 °C. After washing with TBS-T, the blots were incubated with HRP-conjugated secondary antibody (Santa Cruz) in TBS-T containing 5% (w/v) skim milk for 1 h at room temperature. After washing with TBS-T, signal was detected using ECL Prime Western blotting detection reagent (GE Healthcare) with an LAS-4000mini (GE Healthcare).