Complete inhibitor cocktail

Cell pellets were suspended with RIPA lysis buffer with complete inhibitor cocktail (Roche, Basel, Switzerland), quantified with Bradford reagent (Sigma-Aldrich, St Louis, MO, USA), run on standard 10% SDS PAGE gels and transferred to polyvinylidene fluoride (PVDF) membranes for immunoblot (IB). The following antibodies were used for IB: β-actin (Santa Cruz Biotechnology, Dallas, Texas, USA; cat#47778), cathepsin S (Abcam, Cambridge, UK cat#134157), cathepsin B (Abcam cat# ab58802); Secondary antibodies: Anti-Mouse-alkaline-phosphatase (AP)-conjugated (Sigma-Aldrich, cat#, A3562), Anti-rabbit-AP conjugated (Sigma-Aldrich, cat#, A3687). All IBs were scanned at high resolution and band intensity was determined using Image J software (NIH, USA). After subtracting the background, each band was normalized to the corresponding housekeeping protein (that is, β-actin) appearing on the blot. Band intensity was presented in arbitrary units (A.U.) adjacent to the representative IB.

Cells were lysed in lysis buffer (5 mMTris-HCl (pH 7.5), 1 mM EGTA, 250 mM saccharose, 1% Triton X-100) supplemented with complete inhibitor cocktail (Roche Applied Science, Penzberg, Germany) and 10 mM 1,10-phenanthroline monohydrate. Automated Western was performed with equal concentrations of protein per sample using WES™ (Protein Simple, San Jose, CA, USA) according to the manufacturer’s instructions. Detection of CD137 in HT29 and HEK cells via Simple Western is shown in Supplementary Figures S1 and S2 using anti-tGFP antibodies.

Cells were washed once with PBS and lysed in lysis buffer (5 mM Tris–HCl (pH 7.5), 1 mM EGTA, 250 mM saccharose, and 1% Triton X-100) supplemented with cOmplete Inhibitor Cocktail (Roche Applied Science) and 10 mM 1,10-phenanthroline monohydrate to prevent ADAM17 autocleavage (22 (link)). In addition, sonification of cell lysates was performed. Equal amounts of protein were loaded on 10% SDS–PAGE gels. The samples were electrotransferred onto polyvinylidene difluoride membranes (Hybond-P; Amersham) and blocked overnight with 5% skim milk in TBS. After incubation with the indicated antibody in blocking buffer, the membranes were washed three times in TBST (TBS containing 0.1% Tween-20). Primary antibodies were detected using affinity-purified peroxidase-conjugated secondary antibodies (1:10.000) for 1 h at room temperature. Detection was carried out using the ECL detection system (Amersham). Signals were recorded by a luminescent image analyser (Fusion FX7 imaging system; PEQLAB Biotechnologie). To analyse the expression of different antigens on the same polyvinylidene difluoride membrane, Western blots were incubated in stripping reagent (100 mM 2-mercaptoethanol, 2% [wt/vol] SDS, 62.5 mM, and Tris–HCl, pH 6.7) at 55°C for 30 min and re-probed.

Cells were washed once with PBS and lysed either in CHAPS {3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate} lysis buffer containing 50 mM Tris (pH 7.5), 140 mM NaCl, 5% glycerol, 1% CHAPS, 2 mM EDTA, and 40 mM glycerophosphate or in 8 M urea buffer, both containing protease and phosphatase inhibitors (Roche cOmplete inhibitor cocktail). Cell lysates were clarified by centrifugation, separated by lithium dodecyl sulfate (LDS)-PAGE, and electrophoretically transferred to nitrocellulose membranes using the iBlot transfer system (Invitrogen, Life Technologies). Membranes were blocked in Li-Cor Odyssey blocking buffer at a 1:1 ratio with Tris-buffered saline (TBS) for 1 h at room temperature before incubation with the indicated antibodies overnight at 4°C. After washes with TBS containing 0.1% Tween 20, immunoreactive proteins were detected with Li-COR DyLight secondary antibodies and visualized on the Li-COR Odyssey system.

U-2 OS and U-2 OS GFP-RPA2 WT and S/T→D cells were washed twice with PBS and directly lysed on ice in 500 µl TNE buffer (50 mM Tris-HCL pH 8.0, 150 mM NaCl, 0.1% Igepal CA630, 1 mM EDTA) supplemented with 2 mM MgCl₂, cOmplete inhibitor cocktail (Roche), phosphoSTOP (Roche), and 25 U/ml benzonase. Cell lysates were incubated for 5 minutes at room temperature and then centrifuged at 15,000g for 15 minutes. Then, 600 µg of cell lysate was incubated with 0.8 µg of rabbit anti-GFP antibody (Torrey Pines biolabs, TP401) for 3 hours at 4 °C. A 20-µl slurry of protein G-sepharose beads (GE Healthcare, 17-061801) was added per sample for a 1-hour incubation period at 4 °C. The beads were collected by centrifugation, washed five times in TNE buffer, and eluted by boiling in 10× SDS–PAGE loading buffer. Samples were subjected to SDS–PAGE and immunoblotting.

Cells were extracted using a RIPA (radioimmunoprecipitation assay) buffer (Thermo Fisher Scientific) supplemented with Complete Inhibitor Cocktail (Roche Applied Science, Indianapolis, IN). For secretion protein analysis, ~2.5 × 10⁶ cells were seeded in a T25 flask and grown to ~90% confluency. The supernatant media were replaced with serum-free DMEM for 18 h (4 mL) and then collected and concentrated to ~200 µL using an Amicon Ultra 10k MWCO centrifugal filter (Millipore Sigma, Burlington, MA, USA). Protein (~25 µg) from cell lysate or media concentrate was loaded on the SDS-PAGE gels and analyzed by Western blotting, as previously described [7 (link)]. CPE-WT and CPE-ΔN were detected using monoclonal antibody (1:5000 dilution, BD Biosciences). Anti-CXCR2 antibody (1:500 dilution) was from Abcam (Cambridge, MA, USA), and β-actin (1:5000 dilution) was from Cell Signaling Technology (Danvers, MA, USA).

Proteins from cells were extracted with RIPA lysis and extraction buffer (Thermo Fisher, Waltham, MA) supplemented with Complete Inhibitor Cocktail (Roche Applied Science, Indianapolis, IN). 20 μg of the protein from cell lysate was loaded per lane on SDS-PAGE gel and subjected to Western blotting according to our procedure published previously.
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Antibodies against GAPDH (1:5000 dilution) and anti-cyclin D1 (1: 500 dilution) were purchased from Cell Signaling Technology (Danvers, MA).