Alexa fluor 680 dye

MEFs were plated at different densities to reach full confluency at 24, 48, 72 or 96 h post OHT treatment (with the exception of Figure 5E). Lysates were separated at 80 V on 10% acrylamide gel for 2 h. Transfer was carried out at 20 V for 1.5 h using Bolt Mini Blot Modules (Life Technologies) to Immobilon-FL (Millipore) membranes. After blocking in Odyssey blocking buffer (LI-COR) for 30 min, the membranes were incubated overnight with 1:1000 mouse monoclonal anti-Viperin (MaP.VIP | #MABF106) (Millipore), rabbit anti-mouse p56 (gift from Ganes Sen, Cleveland Clinic, Cleveland, OH, USA), mouse monoclonal anti-β-tubulin (TU-06 | ab7792) (Abcam) or monoclonal rabbit anti-STING (D2P2F | #13647) (Cell Signaling). Conjugated secondary with Alexa Fluor^® 680 dye (Life Technologies) or IRdye800 (Rockland) was subsequently used to image the proteins at 700 or 800 nm with an Odyssey scanner (LI-COR).

MEFs were seeded in 24-well plates at a density of ∼25 000 cells per well for 48 h and 50 000 cells per well for 24 h. Analyses were carried out as previously described (18 (link)). Cell lysates were separated at 80 V on 10% acrylamide gels for 2 h. Following transfer at 20 V for 1.5 h using Bolt Mini Blot Modules (Life Technologies) to Immobilon-FL (Millipore) membranes, membranes were blocked 30 min in Odyssey blocking buffer (LI-COR). The membranes were subsequently incubated overnight with 1:1000 mouse monoclonal anti-Viperin (MaP.VIP | MABF106, Millipore), rabbit anti-mouse p56 (gift from G. Sen, Cleveland Clinic, Cleveland, OH, USA), rabbit anti-mouse Mda5 (D74E4 | 5321, Cell Signaling Technology) or mouse monoclonal anti-β-tubulin (TU-06 | ab7792, Abcam). Finally, conjugated secondary antibodies with Alexa Fluor^® 680 dye (Life Technologies) or IRdye800 (Rockland) were used to image the proteins at 700 or 800 nm with an Odyssey scanner (LI-COR).

Tissues were homogenized in T-PER Tissue Protein Extraction Reagent (Thermo Fisher #78510) containing protease and phosphatase inhibitors (Thermo Fisher #78441). After centrifugation at 12,000 g for 15 min, 10 μg of protein (for CD31 analysis) or 30 μg of protein (for SOX2 and NANOG analysis) were loaded on 4-12% gradient Bis-Tris Bolt Plus gel (Thermo Fisher) and transferred to a nitrocellulose membrane (Nitrocellulose Regular Stacks; Novex #IB23001) using an iBlot 2 Gel Transfer Device. Membranes were blocked for 1 h at room temperature in 5% nonfat dry milk in PBS. Then, they were incubated with antibodies against CD31 (1:250; Abcam, ab28364), SOX2 (1:500; Cell Signaling, D6D9) and NANOG (1:500; Cell Signaling, D73G4) diluted in 1X PBS, 0.1% Tween-20 with 5% BSA at 4°C overnight and GAPDH (1:5000; Invitrogen 437000) at room temperature for 1 h. For detection, membranes were incubated with a secondary antibody anti-rabbit or anti-mouse conjugated with Alexa Fluor 680 dye (1:5000; ThermoFisher Scientific) at room temperature for 1 h and imaged on Licor Odyssey scanner.