Phosstop

TUBE2 (UM402; LifeSensors) agarose beads were used to capture polyubiquitin moieties from HCT116 p53+/+, HCT116 p53-/-, and U2OS cell lysates. Cells were harvested in TENT buffer (50 mM Tris-HCl pH 8.0, 2 mM EDTA pH 8.0, 150 mM NaCl, 1% Triton X-100; Sigma-Aldrich) complemented with 1xPIC (Roche), 20 μM PR-619 DUBi (Calbiochem), and 1x PhosSTOP (Roche), incubated on ice for 10 min, then sonicated 12x 30 sec ON/ 30 sec OFF in Bioruptor Pico sonicator (Diagenode). Afterwards, samples were centrifuged at 14,000 g for 10 min at 4°C. Protein concentration of the supernatants was measured with Pierce^TM BCA Protein Assay Kit (Thermo Fisher Scientific). 20 μl TUBEs beads/ IP were washed twice with TBST (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.1% Tween 20; Sigma-Aldrich) complemented with 1xPIC (Roche), 20 μM PR-619 DUBi (Calbiochem), and 1x PhosSTOP (Roche). Between each washing step, beads were centrifuged at 1,000 g for 1 min at 4°C. 1.5 mg protein was added to 20 μl TUBEs beads and exceeded with TENT + inhibitors up to 500 μl final volume, then samples were rotated for 2 h at 4°C. Beads–polyubiquitin complexes were washed three times with TBST + inhibitors, then eluted in 40 μl final volume of the mixture of TENT + inhibitors and NuPAGE^TM LDS Sample Buffer (4x) (Thermo Fisher Scientific) at 100°C for 10 min.

Cortical neurons (DIV16) were washed once with ice cold 1X PBS pH7.4 (Gibco, 10010–023) and then lysed in radioimmunoprecipitation assay buffer (RIPA) buffer (20 mM Tris–HCL pH 8.0, 1 mM NaEDTA pH8.0, 0.5 mM NaEGTA pH 8.0, 1% Triton X-100, 150 mM NaCL, 1X protease inhibitors EDTA-free, 1X PhosSTOP (Roche)) to obtain total cell lysates (TCL). Neuro-2a cells were lysed in polyribosome lysis buffer (PLB) (20 mM Tris–HCL pH7.4, 5 mM MgCl2, 100 mM KCl, 1% NP-40, 1 mM DTT, 20U/µl SUPERase Inhibitor, 1X protease inhibitors EDTA-free, 1X PhosSTOP (Roche)), then subcellular fractionation of lysates was performed by centrifugation (10,000 × g, 10 min, 4 °C) to obtain supernatant (S1) and pellet (P1) fractions. The P1 fraction was then resuspended in RIPA buffer. All protein extracts were prepared in 1X Laemmli buffer and boiled (5 min, 95 °C) for western blot analysis. Samples were resolved on SDS–polyacrylamide gels and transferred to nitrocellulose membranes followed by standard western blotting procedure as described in [69 ]. Membranes were imaged using the LI-COR Odyssey imaging system. Analysis of signal intensity was done using Image Studio Lite Software Version 5.2. Quantification of FUS expression in the subcellular fractions was normalized to GAPDH for TCL and S1 and ponceau red for P1.

For total lysates, cells were lysed in RIPA-buffer (50 mM Tris, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP-40, 1× protease inhibitors (Roche) and 1× PhosStop (Roche)) or MCL-buffer (50 mM Tris, 150 mM NaCl, 1% NP-40, 1× protease inhibitors l(Roche) and 1× PhosStop (Roche)) for 30 min on ice. After clearance by centrifugation at 20,000 × g for 10 min at 4 °C, lysates were boiled in sample buffer (200 mM Tris-HCL, 6% SDS, 20% Glycerol, 0.1 g/ml DTT and 0.01 mg Bromophenol Blue). Proteins were separated by SDS-PAGE and transferred onto nitrocellulose membranes. Membranes were blocked in 5% milk or 5% BSA in TBS supplemented with 0.1% Tween-20 (TBS-T) respectively. Membranes were incubated with primary antibodies overnight at 4 °C in blocking buffer, washed with TBS-T, incubated with secondary HRP-conjugated antibodies for 1 h at room temperature, washed again with TBS-T and immediately analyzed by enhanced chemiluminescence.

Whole-cell extracts were obtained from HeLa cells by extraction in RIPA buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% IGEPAL CA-630, 0.5% sodium deoxycholate, 1 mM EDTA, 1 mM PMSF, 1 mM sodium orthovanadate, PhosSTOP (04906845001, Roche). For chromatin fractionation, cells were harvested from plates, washed with PBS, and resuspended in cytoskeleton (CSK) buffer (10 mM HEPES [pH 7.4]; 300 mM sucrose; 100 mM NaCl; 3 mM MgCl₂; 0.5% Triton X-100; 1 mM PMSF; 0.1 mM sodium orthovanadate; PhosSTOP [04906845001, Roche]; and 1 μg/mL each of leupeptin, aprotinin, and pepstatin) for 15 min on ice. Tubes were centrifuged at 5,000 × g for 5 min at 4°C, and the non-chromatin bound supernatant was frozen in liquid nitrogen. The chromatin pellet was washed twice using CSK buffer without Triton X-100, centrifuged at 5,000 × g for 5 min at 4°C, and frozen in liquid nitrogen. Protein was prepared from chromatin pellets by extraction in RIPA buffer.

The cytosolic or nuclear extracts were prepared as previously described [19 (link)]. Briefly, DMSO- or CA-treated cells were dissolved in hypotonic buffer (10 mM Hepes-KOH (pH 7.9)/10 mM KCl/0.1 mM EDTA) supplemented with a protease inhibitor cocktail and PhosSTOP (Roche, Mannheim, Germany), and NP-40 (Wako, Osaka, Japan) was then added to a final concentration of 0.6%. After centrifugation, the supernatants were stored as the cytosolic fraction, and the separated nuclear pellets were used as the nuclear fraction preparation. To prepare whole cell lysate, the cells were then directly lysed with RIPA buffer (50 mM Tris-HCl (pH 8.0)/150 mM NaCl/1% NP-40/0.5% Na-DOC/0.1% SDS) supplemented with protease inhibitor cocktail and PhosSTOP (Roche, Basel, Switzerland). The protein concentration was determined using a BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA).

Before phosphorylation, recombinant SYNJ2BP (WT or S21A, 0.5 µg) was dephosphorylated using 1 µl of CIP (NEB) in 9 µl 1× CutSmart buffer. The reactions were incubated at 37 °C for 1 h and stopped by 1× PhosStop (Roche). In vitro phosphorylation of dephosphorylated recombinant SYNJ2BP (0.5 µg in 10 µl) was performed in kinase reaction buffer (8 mM MOPS/NaOH, pH 7 and 200 µM EDTA) with 500 µM ATP (Serva), 200 µM AMP (Serva) and active recombinant AMPK (16 ng) (Sigma-Aldrich, 14-840) in a total volume of 30 µl. Alternatively, neuronal lysates were prepared from cells by Dounce homogenization in lysis buffer (20 mM Tris/HCl, pH 7.2, 30 mM NaCl, 10 mM MgCl₂, 10% glycerol, 1 mM EDTA, 200 µM PMSF, protease inhibitor cocktail (Roche) and PhosStop (Roche)) and cleared by centrifugation at 2,300g for 1 min at 4 °C. The supernatant was collected and immediately used for in vitro phosphorylation assays or snap-frozen. Then, 0.5 µg SYNJ2BP was mixed with 25 µl cytosolic extract supplemented with 500 µM ATP in a final volume of 40 µl. The samples were incubated at 30 °C for 2 h (shaking). The reactions were stopped by addition of Laemmli sample buffer at 95 °C for 5 min.