Easypack protease inhibitor cocktail

Tumor tissues were mechanically homogenized in lysis buffer (2% SDS, 100 mM Tris-HCl, pH 7.8) supplemented with protease inhibitors (Complete™ ULTRA Tablets, Mini, EASYpack Protease Inhibitor Cocktail, Roche, USA), using a POLYTRON® PT 1200 and sonicated for three cycles at 30% amplitude (20 s bursts with 20 s pauses). Samples were centrifuged at 16,000 x g for 10 min at 4 °C to remove the tissue debris. The supernatants were collected and the protein concentration was measured by using a BCA protein assay (Pierce™ BCA Protein Assay Kit, Thermo Scientific, USA). Aliquots of SDS-lysates containing 200 µg of total protein from each sample were processed according to the filter-aided sample preparation (FASP) method, using Microcon 10 kDa centrifugal filter units (Merck, USA) operated at 10,000 x g for 50 min at 20 °C^{96 (link)–98} . Next, trypsin/LysC Mix (Promega, USA) was added to the filters at an enzyme-to-protein ratio of 1:100 (w/w), and incubated for 12 h at 37 °C; a second digestion was carried out with trypsin (Promega, USA) at an enzyme-to-protein ratio of 1:100 (w/w) at 37 °C for 4 h. Following protein digestion, peptides were filtered through the membrane and purified with reversed-phase chromatography using C18 micro-pipette tips (TopTip^TM, PolyLC inc, USA), according to the manufacturer’s instructions. Peptides were dried in a vacuum concentrator and stored at −20 °C.

Cells were lysed with 1x Laemmli Sample buffer (60 mM tris(hydroxymethyl)aminomethane pH 6.8, 10% Glycerol, 2% Sodium dodecyl sulfate, 1.5% 2-mercaptoethanol, 1.5% Dithiothreitol, and 0.005% bromophenol blue) containing phosphatase and protease inhibitors (PhosSTOP and cOmplete ULTRA tablets, mini, EASYpack protease inhibitor cocktail, Roche), and proteins were analyzed by Western blot. To evaluate STAT1 and STAT2 activation, we measured their phosphorylation using a rabbit anti-phospho-STAT1 (cs9167, Cell Signaling, 1:1000) and a rabbit anti-phospho-STAT2 antibody (cs8841, Cell Signaling, 1:4200). Immunoreactive bands were detected using ChemiDoc XRS+ (Bio-Rad) after incubation with a secondary anti-rabbit antibody coupled with the horseradish peroxidase (HRP, 1:5000) and exposure to the SuperSignal West Femto chemiluminescent substrate (Thermo Fisher Scientific). Densitometric quantification of the bands was performed with ImageLab software (Bio-Rad). Data were normalized for the expression of the housekeeping protein β-actin (cs4967, Cell Signaling, 1:5000).

Briefly, infiltrated 7 dpi whole-leaf samples were homogenized using a Matstone 6-in-one juice extractor (Matstone, PH), protein were extracted using PBS, pH 7.4 containing cOmplete™ ULTRA tablets mini, and EASYpack protease inhibitor cocktail (Roche, CH). The supernatant was clarified by a series of centrifugation and filtration steps. The bNAbs were then purified by protein A affinity chromatography using a HiTrap^® Protein A High-Performance column (Cytiva, USA), coupled to a Äkta Avant 150 (Cytiva, USA), as per the manufacturer's instructions.

For total protein isolation, colonic biopsy samples were minced into small pieces with a razor blade in a Petri dish and were transferred into 1 ml RIPA lysis buffer [Millipore, 20-188]. The 10 × RIPA lysis buffer was diluted in AccuGene water [Lonza, 51200] and completed with EASYpack Protease Inhibitor Cocktail [Roche, 05892970001]. The biopsy samples were homogenised with a Branson Sonifier SFX150 on ice [four cycles, 10 s sonication, and 10 s break per cycle]. After that, the samples were centrifuged at 3500 rpm for 10 min at 4°C. The supernatant was transferred into a new 1.5 ml tube, snap-frozen, and stored at -80°C until use. For total protein isolation from stool samples, 100 mg stool was vortexed in 300 µl RIPA lysis buffer with protease inhibitor for 5 min. After that, the samples were centrifuged at 4000 g for 10 min at 4°C. The supernatant was transferred to a new, sterile tube, and the centrifuge step was repeated at 12000 g for 10 min at 4°C. The supernatant was transferred into a new 1.5 ml tube, snap-frozen, and stored at -80°C until use.

(Knockout) cells were grown in a 6-well plate until confluent, washed twice with cold PBS, and lysed in RIPA lysis and extraction buffer (Thermo Fisher Scientific) supplemented with phosphatase inhibitors (PhosSTOP EASYpack; Roche) and protease inhibitors (EASYpack protease inhibitor cocktail; Roche). After 5 min of incubation at 4°C, cells were scraped, transferred into Eppendorf tubes, and incubated in a tube revolver (Thermo Fisher Scientific) for 1 h at 4°C. Samples were centrifuged for 15 min at 15,000 × g at 4°C. The supernatants were diluted in NuPAGE LDS sample buffer (Supplier: Thermo Fisher Scientific) (4×) and analyzed by Western blot analysis as described previously (45 (link)). Monoclonal antibodies targeting RIG-I (AG-20B-0009-C100; Bio-Connect), PKR (ab226819; Abcam), and MAVS (ab89825; Abcam) were used according to the manufacturer’s instructions. Secondary polyclonal rabbit anti-mouse IgG-horseradish peroxidase (HRP) (P0260; Agilent) and polyclonal swine anti-rabbit IgG-HRP (P0217; Agilent) antibodies were used at a 1:1,000 dilution. Imaging was performed using an Amersham imager 600 (GE Healthcare).

Cells and tumors were lysed in RIPA buffer supplemented with protease and phosphatase inhibitors (cOmplete ULTRA Tablets, Mini, EASYpack Protease Inhibitor Cocktail; and PhosSTOP, both from Roche). Total lysates were quantified by BCA (Pierce BCA Protein Assay Kit, Thermo Fisher Scientific), resolved by SDS-PAGE, and transferred to nitrocellulose membranes. After blocking with 5% (wt/vol) nonfat dry milk in TBS-Tween, membranes were incubated with the corresponding antibodies (Supplemental Table 2) overnight at 4°C. Secondary antibodies (Supplemental Table 2) were chosen according to the species of origin of the primary antibodies and detected by an enhanced chemiluminescence system (Bio-Rad). Densitometric analysis of the relative expression of the protein of interest versus the corresponding control was performed with Fiji ImageJ software. Complete unedited blots can be found in the supplemental material.