Complete mini

Colon tissues were homogenized and lysed on ice in a buffer containing 0.5% NP-40, 40 mM Tris-HCl (pH 8.0), 120 mM NaCl, phosphatase inhibitor cocktail (PhosSTOP, Roche Applied Science, Indianapolis, IN), and a protease cocktail inhibitor (Complete Mini, Thermo Fisher Scientific Inc., Waltham, MA). Protein levels in the lysate were measured with the modified bicinchoninic acid method (Thermo Fisher Scientific Inc.). Proteins were denatured with sodium dodecyl sulfate sample buffer at 95 °C for 5 min and then subjected to 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a PVDF membrane. Membranes were blocked in a Tris-buffered saline buffer (10 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.1% Tween-20) containing 5% skim milk and then incubated overnight with one of the antibodies (IL-1β, IL-18, caspase-1, NLRP3 and CDX2) (Supplementary Table 2). Bound antigen-antibody complexes were detected with the appropriate secondary antibodies coupled to HRP with enhanced chemiluminescence according to the manufacturer’s instructions (Amersham, Arlington Heights, IL). The membranes were stripped and re-probed with mouse anti-β-actin (Sigma-Aldrich Co.) as a loading control. Relevant bands were quantified with laser-scanning densitometry (Image Quant LAS 4000 mini, Image Quant TL Analysis Toolbox, GE Healthcare UK Ltd).

Treated cells were lysed in EBC buffer (50 mM Tris pH 8.0, 120 mM NaCl, 0.5% NP-40) supplemented with protease inhibitor cocktail (Complete Mini, Thermo) and phosphatase inhibitors (phosphatase inhibitor cocktail set I and II, Bimake) and were collected into 1.5 mL EP tube when they reached to 100% confluency in 6 or 10 cm dishes. For IP, 1 mg lysates were incubated with the appropriate indicated antibody (1–2 μg) and ProteinA Sepharose beads (Sigma) overnight at 4 °C. Immuno-complexes were then washed four times with NETN buffer (20 mM Tris, pH 8.0, 100 mM NaCl, 1 mM EDTA and 0.5% NP-40). After final spin, the beads were boiled with 50 μl of 2 × SDS Laemmli buffer for 15 min, and the samples were subjected to IB. For IB analysis, cells were collected and total protein lysed with EBC buffer described above. After boiling with SDS laemmli buffer, equal amount of protein was separated by SDS-PAGE gel and immunoblotted with standard protocols. Antibodies used in this study are as follows: OTUD6A (Invitrogen, PA5-62772, 1:500; Proteintech, 24486-1-AP, 1:1000), Brg1 (CST, #72182, 1:1000), AR (Santa Cruz, sc-7305, 1:500), PTEN (CST, #9556, 1:1000), HA (Proteintech, 51064-2AP, 1:5000), Flag (Sigma, F3165, 1:1000), GFP (Proteintech, 66002-1-Ig, 1:5000), Myc (Abmart, M20002S, 1:3000), Ub (Santa Cruz, sc-8017, 1:500), β-Tubulin (BPI, AbM59005-37B-PU, 1:5000).

Lysates of mice skeletal muscle tissue generated under the addition of proteinase inhibit cocktail Complete Mini (Thermo, Waltham, MA, USA) and phosphatase inhibitor cocktail PhosSTOP (Thermo, Waltham, MA, USA). The total protein of the lysates was measured by the Pierce BCA Protein Assay Kit (Thermo, Waltham, MA, USA). Co-immunoprecipitation (co-IP) was completed using the Thermo Scientific Pierce co-IP kit (#26149) following the manufacturer’s protocol. Ten micrograms of the antibody were incubated with the delivered resin and covalently coupled. The antibody-coupled resin was incubated with 200 µL of the whole mice skeletal muscle protein lysates overnight at 4 °C, respectively. The resin was washed, and the protein complexes bound to the antibody were eluted. Subsequent Western blot analyses were performed as described before.

Following anesthesia, the brains were removed from the skull and these were dissected into the right or left hemispheres. For protein extraction, they were placed in 10 volumes of cold homogenization buffer (120 mM NaCl, 50 mM Tris, pH 7.4) with protease inhibitors (Complete Mini, Gibco, Grand Island, NY, USA) being freshly added. Tissue then was homogenized by sonicator. Concentrations of protein were checked by the Bradford method (BioRad, Richmond, CA, USA). By adding the sampling buffer, equal amount of protein, 20 μg, was loaded and separated. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used with 10% polyacrylamide and 0.05% bis-acrylamide [11 (link)]. By separating proteins on the gels, they were transferred into nitrocellulose membrane. They were probed with anti-NT-3 (1 : 300, Santa Cruz, CA, USA) and anti-trkC (1 : 300, Santa Cruz, CA, USA) as primary antibody. For secondary antibody, Peroxidase anti-rabbit IgG (Vector, PI-1000, 1 : 3000 dilution) was used. As an internal control, anti-β tubulin (1 : 300, Santa Cruz, CA, USA) was checked on the sample membrane. We detected signals with enhanced chemiluminescence (Supersignal, Pierce, Rockford, IN, USA), using autoradiogram by exposing 10 to 30 min [6 (link)–8 (link)].

Total proteins were extracted with RIPA buffer, to which protease inhibitors (complete Mini, GIBCO, Grand Island, NY) had been freshly added. Protein concentration was determined by the Bradford method (Bio-Rad, Richmond, CA). Protein extracts (20 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Protein separation was performed in 10% polyacrylamide with 0.05% bis-acrylamide and transferred to a nitrocellulose membrane. Anti-PARP (1:1000 dilution; Cell Signaling Technology, Berverly, MA), anti-Bax (1:1000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA), anti-pAkt (1:1000 dilution; Cell Signaling Technology, Berverly, MA), and anti-pERK (1:1000 dilution; Cell Signaling Technology, Berverly, MA) were used as primary antibodies. Anti-tubulin antibody (1:2000 dilution; NeoMarkers) was used for standardization of the protein amount. Immunoreactivity was detected by enhanced chemiluminescence (Supersignal, Thermo Scientific, Rockford, IL).