Anti gapdh

SDS-PAGE and Western blotting were performed as previously described (45 (link)). In brief, at 2 days postinfection, cells were washed with phosphate-buffered saline (PBS) and lysed with coimmunoprecipitation (Co-IP) buffer. The samples were separated on 4% to 12% Bis-Tris gradient acrylamide gels (Invitrogen) and blotted onto polyvinylidene difluoride (PVDF). Blotted membranes were probed with anti-ZAP (GeneTex, catalog no. GTX120134) diluted 1 to 1,000 or with anti-HA tag (catalog no. C29F4; Cell Signaling) diluted 1 to 2,000, anti-KHNYN (Santa Cruz Biotechnology, catalog no. sc-514168) diluted 1 to 50, or anti-TRIM25 (BD Biosciences, catalog no. 610570) or with anti-GAPDH (BioLegend, catalog no. 607902) or anti-beta-actin (abcam, catalog no. ab8227), each diluted 1 to 2,000, or anti-ISG15 (Santa Cruz Biotechnology, catalog no. sc-166755) diluted 1 to 1,000. Proteins were also stained using anti-ACE2 (abcam, catalog no. ab15348) and anti-SARS-CoV-2 Spike (Biozol, catalog no. GTX-GTX632604) primary antibodies, both diluted to 1 to 1,000. Subsequently, blots were stained with IRDye 680RD goat anti-rabbit IgG (H+L) (Li-Cor, catalog no. 926-68071), IRDye 800CW goat anti-mouse IgG (H+L), and IRDye 800CW goat anti-rat IgG (H+L) (Li-Cor, catalog no. 925-32219) secondary antibodies, all diluted 1 to 20,000, and were scanned using a Li-Cor Odyssey reader.

2D cultures and 3D constructs were lysed with RIPA buffer (R0278, Merck) containing protease inhibitor cocktail (11836153001, Roche, Basel, Switzerland). Total protein content was quantified with a BCA protein assay kit (39228, Serva, Heidelberg, Germany) and 5 or 10 μg of protein were loaded into 8% polyacrylamide gels and run by applying 225 V for 40 min. Afterwards, proteins were transferred to a PVDF membrane (IPVH07850, Merck) by applying 40 V for 2 h. The membrane was then blocked for 1 h with 4% (w/v) nonfat powdered milk in 0.2% PBS-Tween. Next, the membrane was incubated with primary antibodies anti-EGFR (700308, Invitrogen) at 1:1000 and anti-GAPDH (649201, Biolegend, San Diego, CA, USA) at 1:2000 for 1 h at room temperature. The membrane was then washed and incubated with secondary antibodies anti rabbit-HRP (ab6721, abcam) and anti mouse-HRP (ab6820, abcam), both at 1:1000 for 1 h at RT. Finally, the membrane was revealed for HRP detection with a SuperSignal West Pico Chemiluminescent Substrate (34080, Thermo Fisher Scientific, Waltham, MA, USA). Chemiluminescent images were taken in the ImageQuant^TM LAS 4000 mini (GE HealthCare, Chicago, IL, USA). Protein bands were quantified using ImageJ software and expressed as a ratio between the protein of interest and the loading control. Each blot was repeated three times (N = 3).

Skeletal muscles were homogenized in one volume of Tris-EDTA buffer pH 7.4 with 1 Mm phenylmethyl-sulfonyl fluoride (PMSF) using Ultraturrax T25 (Janke & Kunkel IKA Werk, Staufen, Germany). Then, the same volume of buffer containing 2% glycerol, 4% SDS, and 0.125 M Tris pH 6.8 was added to the homogenates and mixed. Muscle homogenates were incubated at 50 °C for 20 min and centrifuged at 14,000 rpm to separate insoluble material. Protein supernatant content was determined using BCA Assay Kit (Thermo Fisher) with BSA as the standard. Muscle extract aliquots (20–40 µg) were subjected to SDS-PAGE and transferred onto PVDF membranes (Thermo Fisher). Membranes were blocked in 5% BSA in TBS (50 Mm Tris-CL, pH 7.6; 150 Mm NaCl) and then incubated at 4 °C overnight with: anti-Fibronectin (F3648, Sigma Aldrich), anti-nTyr (N0409, Sigma), anti-Atrogin-1 (PA5-106917, Invitrogen), anti-CTGF (D8Z8U, Cell Signaling, Danvers, MA, USA), anti-GAPDH (631402, Biolegend, San Diego, CA, USA), anti-nNOSµ (617000, Invitrogen), anti- TGF-β3 (D-B3, DSHB). Membranes were incubated with HRP-conjugated secondary antibodies and visualized by enhanced chemiluminescence (Cyanagen, Bologna, Italy) in ImageQuant LAS 500 equipment. Densitometric analysis and quantification were performed using ImageJ software (NIH).

HEK293 cells were transfected with a FLAG-N expression plasmid by using Lipofectamine 2000 (Invitrogen). At 24 hpi, cells were harvested by trypsinization and centrifugation. After two washes with PBS, cells were swollen with Hypotonic Lysis Buffer (20 mM HEPES pH 7.4, 10 mM NaCl, 3 mM MgCl₂, and 1× Protease inhibitor (Gold Biotechnology)) and lysed with 27G needle in presence of 25 μg/mL digitonin. Digitonin is a mild detergent that has been shown not to lyse nuclei [38 (link)]. Insoluble, nuclear fraction was pelleted by centrifugation for 10 min at 1500× g, washed with PBS, and lysed with NuPAGE LDS Sample Buffer (Invitrogen). Soluble, cytoplasmic fraction was spun down for 15 min at 10,000× g, and the clear supernatant was kept for analysis. Samples were electrophoresed under reducing and denaturing conditions, and transferred onto a PVDF membrane overnight. Antibodies used for the blotting were anti-FLAG (Sigma, F3165, 1:2000), anti-GAPDH (BioLegend, 607901, 1:1000), anti-histone H2B (BioLegend, 606301, 1:1000), anti-mouse (Sigma, A2554, 1:6000), and anti-rat (BioLegend, 405405,1:2000).

Isoproterenol hydrochloride, propranolol (Pro), and Griess reagent were purchased from Sigma-Aldrich (St. Louis, MO, USA). Halt protease and phosphatase inhibitor cocktail were purchased from Pierce Biotechnology (Rockford, IL, USA). The antibody against HO-1 was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA); secondary anti-rabbit IgG (H and L, horseradish peroxidase-linked) was purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-GAPDH was purchased from BioLegend Inc. (San Diego, CA, USA) and the anti-SOD1 and SOD2, GPx, catalase, and Nrf2 antibodies were from Abcam (Cambridge, UK). A multiple ELISA kit was obtained from Merck Millipore (Darmstadt, Germany). Zoletil was obtained from Virbac Laboratories (Fort Worth, Texas, USA). Novolink™ Polymer Detection System was purchased from Leica (Newcastle, UK). All other chemicals were of analytical grade, including Avicel PH 102 (FMC Biopolymer, USA) and Ludiflash (Pharma Ingredients & Services, Bishop, Texas, USA).

Cell lysates were prepared using 1 × RIPA buffer (EMD Millipore) consisting of 50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 0.25% deoxycholic acid; 1% NP-40; 1 mM EDTA; and freshly added Halt™ Protease and Phosphatase Inhibitor Single-Use Cocktail (Thermo Fisher, Waltham, US). Protein concentrations were measured using a BCA protein assay kit (Thermo Fisher, Waltham, US). The following antibodies were used: anti-STING-pS366 (CST 85735); anti-STING (CST 13647); anti-TBK1-pS172 (CST 5483); anti-TBK1 (CST 3504); anti-NFκb p65-pS536 (CST 3033); anti-NFκb p65 (CST 8242); and anti-GAPDH (Biolegend HRP anti-GAPDH Ab 649203). Horseradish peroxidase-conjugated secondary antibodies (CST) were used to amplify the signal from the primary antibodies.