Phosstop phosphatase inhibitor

Biological replicates were pooled and lysed in RIPA buffer supplemented with cOmplete protease inhibitor cocktail (Sigma) and PhosSTOP phosphatase inhibitor (Sigma) for 45 minutes on a rotator at 4^oC. After spinning at 15,000 rpm for 15 minutes at 4°C, protein lysate was quantified using bicinchoninic acid protein kit and boiled in sample buffer at 70°C for 10 minutes. Samples were run in Bolt 4%–12% Bis-Tris gels (Thermo Fisher Scientific) according to manufacturer recommendations and transferred to PVDF membranes using the Trans-Blot Turbo system (Bio-Rad). Membranes were subsequently blocked in 5% milk (in TBST) for 1 hour at RT and incubated with primary antibody diluted in 5% BSA (in TBST) overnight at 4°C. After 3 washes in TBST, membranes were incubated with a secondary antibody diluted in 5% milk (in TBST) for 1 hour at RT. After another set of 3 washes in TBST, membranes were developed using SuperSignal West Pico Plus chemiluminescence kit (Thermo Fisher Scientific) and imaged with ChemiDoc (Bio-Rad).

The total cell lysate was extracted with lysis buffer, which is a mixture of RIPA buffer (Nacalai Tesque) and PhosSTOP phosphatase inhibitor (Sigma-Aldrich, St. Louis, MO). Western blot analysis was performed as described previously^{39 (link)}. The antibodies and enhanced chemiluminescence solution are described in Supplementary Table 6. Full-length APC was detected with APC antibody (#2504) from Cell Signaling Technology, and truncated APC was detected with APC antibody (sc-9998) from Santa Cruz Biotechnology (Dallas, TX) raised against the N-terminus of APC. β-Actin was used as the loading control.

Cells were seeded in 6-well plates and treated as described above with the exception that TNFα treatments persisted for 10 min. Cell lysates were harvested using NETN buffer (150 mM NaCl, 1 mM EDTA, 20 mM pH 8.0 Tris, 0.5% NP-40) containing 1X cOmplete™ Protease Inhibitor Cocktail without EDTA (PI) (Sigma-Aldrich) and 1X PhosSTOP™ phosphatase inhibitor (Sigma-Aldrich). Protein concentrations were determined using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific) and a SpectraMax Microplate Reader (Molecular Devices, San Jose, CA, USA). Equal amounts of protein were separated on 7.5% SDS polyacrylamide gels, transferred to PVDF membranes which were subsequently blocked for 1 h at room temperature using 5% milk or 5% BSA in 1X TBST depending on primary antibody. Membranes were incubated with primary antibody overnight at 4 °C, washed with 1X TBST, incubated with secondary antibody for 1 h at room temperature, and washed again. Blots were imaged on the Odyssey Fc (LI-COR, Lincoln, NE, USA) system using the chemiluminescent and 700 nm channels for 10 min and 30 sec, respectively. All blots for a given experiment were derived from the same experiment and were processed in parallel. Uncropped and unprocessed scans of all blots presented in this manuscript are provided in the Supplementary Information document. Antibody information can be found in Supplemental Table 5.

Whole lysates were collected by lysing cells in radioimmunoprecipitation assay buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X‐100, 1% sodium deoxychalate, 0.1% SDS) supplemented with cOmplete™, EDTA‐free protease inhibitor cocktail (Sigma) and PhosSTOP phosphatase inhibitor (Sigma). Immunoblotting was performed by electrophoresing equal amounts of protein (determined by bicinchoninic acid assays) through precast BOLT gels (Invitrogen), followed by turbo transfer at 25 V for 9 min onto nitrocellulose membranes (BioRad). Membranes were then blocked in 5% BSA in tris‐buffered saline containing 0.05% TWEEN^® 20, followed by probing with the indicated antibodies (Supporting Information Table S3). Proteins were visualized using Clarity ECL (BioRad) and Chemidoc. Membranes were either stripped using ReBlot Plus Strong Solution (Merck) at RT for 15 min or quenched with hydrogen peroxide 30% (Merck) at 37°C for 20 min, prior to reprobing of blots.

Sequential extraction of αSYN from brain tissue was performed as previously described [37 (link)]. In short, flash-frozen brain tissue samples were homogenized with a PreCellys Evolution (VWR, Stockholm, Sweden) in ice-cold Tris-buffered saline (TBS) supplemented with cOmplete protease inhibitor (Roche, Mannheim, Germany) and PhosStop phosphatase inhibitor (Sigma, Gillingham, UK) at a 1:10 w/v ratio. Next, αSYN species of decreasing solubility were extracted with TBS with 0.5% Triton X-100 (TBS-T) at 16,000× g and 70% formic acid (FA) at 100,000× g.

Cells were plated in complete media, left to adhere overnight and then stimulated for designated periods with specific concentrations of LPS, as indicated in individual figure legends. Media was removed and cells were harvested in chilled phosphate‐buffered saline (PBS) with a cell scraper. Cells were pelleted and lysed in 700 µL co‐immunoprecipitation lysis buffer [20 mM TRIS (pH 7.4), 150 nM NaCl, 1% NP40 and 5% glycerol] supplemented with cOmplete, ethylenediaminetetraacetic acid‐free Protease Inhibitor Cocktail (Sigma‐Aldrich) and PhosSTOP phosphatase inhibitor (Sigma‐Aldrich). Lysates were passed through a 28‐gauge needle and centrifuged at 10 000g for 15 min. 400 µL of lysate was added to columns containing 20 µL of Pierce protein G plus agarose beads (Life Technologies) and 2 µL of capture antibody (Supplementary table 4). Columns were incubated at 4°C while rotating for 1 h. Columns were centrifuged to remove the after‐bind mix, then washed three times with co‐immunoprecipitation lysis buffer. Bound samples were boiled using Bolt LDS Sample Buffer (Life Technologies) and NuPAGE Sample Reducing Agent (Life Technologies) before being eluted from columns and immunoblotted.