Odyssey fc instrument

Western blot analyses were conducted as previously described (3 (link)). Antibodies against the following proteins were used: MLCK (Sigma-Aldrich, M7905; 1:1000), STING (Cell Signaling Technology, 13647S; 1:1000), p-TBK1 (Cell Signaling Technology, 5483; 1:500), TBK1 (Cell Signaling Technology, 3504; 1:500), and SOX9 (Cell Signaling Technology, 82630S; 1:500). HRP-conjugated secondary antibodies were used (Cell Signaling Technology, 7074S and 7076S; 1:10,000) and signals were developed using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, 34095). Images were acquired using an Odyssey Fc instrument and analyzed using Image Studio software (LI-COR Biosciences). Following detection, immunoblots were stripped (Thermo Fisher Scientific, 46430) and reused for detection of total protein levels. See complete unedited blots in the supplemental material.

Cell pellets were lysed for 30 min in ice-cold extract buffer (50 mM TrisHCl), pH 7.4, 250 mM NaCl, 0.1% Nonidet NP40, 5 mM EDTA, 50 mM NaF and a protease inhibitor cocktail (Sigma-Aldrich). Lysates were cleared by centrifuging at 12,000 rpm for 15 min, and the protein concentration was determined using a Bio-Rad assay kit (Bio-Rad Laboratories S.r.l., Hercules, CA, USA). Cell lysates (50 µg) were resolved on 10–12% SDS-PAGE (polyacrylamide gel electrophoresis) gels. Proteins were then transferred to nitrocellulose membranes (Merck Millipore, Burlington, MA, USA). Immunoblotting was conducted with the following antibodies: rabbit anti-γH2AX Cat#9718 (Cell Signaling, Danver, MA, USA, 1:1000) and mouse anti-ACTIN sc-47778 (Santa Cruz Biotechnology, Dallas, TX, USA, 1:500). The secondary antibodies conjugated with horseradish peroxidase (HRP) goat anti-rabbit Cat#170-6515 and goat anti-mouse Cat#170-6516 were purchased from Bio-Rad Laboratories S.r.l. The HRP substrate (ECL Western Blotting Detection, Amersham-Life Science, Amersham, UK) was added, and the signal was detected with the Odyssey Fc instrument (Li-COR, Lincoln, NE, USA). The uncropped version of the gels are shown in Supplementary Figure S6.

Cell pellets were lysed for 30 min in ice-cold whole cell extract buffer (50 mM TrisHCl pH 7.4, 250 mM NaCl, 0.1% Nonidet NP40, 5 mM EDTA, 50 mM NaF and a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). Lysates were cleared by centrifuging at 12,000 rpm for 5 min and the protein concentration was determined using a BioRad assay kit (BioRad, Hercules, CA, USA). Cell lysates (50µg) were resolved on 10–12% SDS-PAGE (polyacrylamide gel electrophoresis) gels. Proteins were then transferred to nitrocellulose membranes (Merck Millipore, Burlington, MA, USA). Immunoblotting was carried out with the following antibodies: rabbit anti-PARP #9542 (cell signaling, 1:1000), rabbit anti-γH2AX #9718 (Cell Signaling, Danver, MA, USA, 1:1000), goat anti-ACTIN sc-1615 (Santa Cruz Biotechnology, Dallas, TX, USA, 1:500) and mouse anti-RAN sc-271376 (Santa Cruz Biotechnology, 1:500). The secondary antibodies conjugated with horseradish peroxidase (HRP) anti-rabbit #1706515 and anti-mouse #1706516 were purchased from BIO-RAD Laboratories S.r.l., anti-goat sc-2354 from Santa Cruz Biotechnology. HRP substrate (ECL Western Blotting Detection, Amersham-Life Science, Amersham, UK) was added and the signal was detected with the Odyssey Fc instrument (Li-COR). All the uncut filter and relative densitometric raw data have been included in Figure S7.

Preparation and Western blot of colon samples homogenates was performed as described previously.⁵³ (link) After transfer, total protein content was assayed using the Revert 700 Total Protein Stain kit (926-11010; LI-COR Biosciences, Lincoln, NE). Specific proteins were detected using commercially available primary antibodies: YAP/TAZ (cat. 8418, 1:1000; Cell Signaling Technology), α-actin (cat. A5228, 1:1000; Sigma-Aldrich), smooth muscle myosin heavy chain (cat. ab53219, 1:1000; abcam), Chrm2 (cat. AMR-002, 1:1000; alomone labs, Jerusalem), Chrm3 (cat. AMR-006, 1:200; alomone labs), SRF (cat. 5147S, 1:1000; Cell Signaling Technology), and myosin light chain (3672S, 1:1000; Cell Signaling Technology). HRP-conjugated or fluorescently labeled secondary antibodies were used and images were acquired using the Odyssey Fc instrument and analyzed using Image Studio software (LI-COR Biosciences).

Frozen tissues were lysed in ice-cold RIPA buffer complemented with 1 × protease inhibitor cocktail (#11836153, Roche, Monza, MB, Italy). Protein concentrations were determined using the Pierce BCA protein assay reagent (#23227, Thermo Scientific). Lysates (30 μg) were separated on NuPage Bis-Tris 4-12% Pre-Cast gels (#NP0322BOX, Invitrogen) and transferred to nitrocellulose membranes (Whatman, Dassel, Germany). Primary antibodies rabbit anti-PGC-1α (1:1000, Chemicon #AB3242) and mouse anti-α-tubulin (1: 10,000, Sigma-Aldrich #T5168) were used with the appropriate secondary antibodies: respectively goat anti-rabbit IgG (H + L) IRDye 800CW (1:2500, #926-32211, LI-COR Biosciences, Bad Homburg, Germany); goat anti-mouse IgG (H + L) IRDye 680RD (1:3500, 926-68070, LI-COR Biosciences). Signals were acquired in dual infrared fluorescence with the Odyssey FC instrument (LI-COR Biosciences). Quantification was performed using Image Studio Lite software. The intensity of the bands was calculated from three immunoblots with samples from different cell preparations. Mean values were referred to as the control value, set at 1.0.

Cell pellets were lysed in the lysis buffer [10 (link)] added with protease and phosphatase inhibitor cocktail for 30 min on ice. Insoluble material was pelleted at 10,000× g for 15 min at 4 °C, and the protein concentration was determined using a BioRad assay kit (BioRad, Hercules, CA, USA). Thirty μg of total cellular proteins were separated on SDS-PAGE and electrotransferred to activated PVDF membrane (Merck Millipore, Burlington, MA, USA). Immuno-blotting was carried out with anti-GLS1 (Cell signaling, 1:1000), anti-GPT2 (Santa Cruz Biotechnology, Dallas, TX, USA 1:250) and anti-RAN (Santa Cruz Biotechnology, Dallas, TX, USA 1:500) primary antibodies and anti-mouse and anti-rabbit peroxidase labelled secondary antibodies (BioRad, Hercules, CA, USA). Horseradish-peroxidase substrate (ECL Western Blotting Detection, Amersham-Life Science, Little Chalfont, UK) was added and the signal was revealed through an Odyssey Fc instrument (LI-COR, Lincoln, NE, USA).

Cells were washed with PBS and lysed with RIPA (Sigma) including 1× HALT protease inhibitor (Life Technologies) for 30 min on ice. Lysates were then cleared by centrifugation at 20,000 rcf for 10 min at 4°C, and protein concentrations were determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). 10–20 µg of protein was separated on a 12–4% Bis-Tris or 20–4% Tris-Glycine polyacrylamide gel. Proteins were transferred to nitrocellulose membranes by standard wet transfer and then briefly washed in Tris-buffered saline (TBS) and blocked in 3% nonfat dry milk (Bio-Rad) in TBS for 1–2 h. The membranes were then washed and incubated with primary antibodies (listed below) in 1% nonfat dry milk in TBS + 0.1% Tween 20 (TBS-T) under shaking at 4°C overnight. Membranes were washed with TBS-T and incubated with secondary antibodies for 1–2 h. Membranes were washed 2× with TBS-T and 2× with TBS, and then fluorescence was detected using a Li-COR Odyssey Fc instrument. Primary antibodies were anti-ADAR: Cell Signaling Technologies 14175; anti-hnRNPC: abcam ab10294; anti-GAPDH: Cell Signaling Technologies 5174; anti–RIG-I: Adipogen AG-20B-0009-C100; anti-MDA5: Enzo Life Sciences ALX-210-935-C100; and anti-UPF1: abcam ab109363; secondary antibodies were Li-COR goat–anti-rabbit IgG or goat–anti-mouse IgG, IRDye 680 RD or IRDye 800 CW conjugates.