Irdye 680lt

Primary antibodies used included the following: mouse anti-FLAG (Sigma), rabbit anti-FLAG (Sigma), mouse anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (EMD Millipore), mouse anti-sorting nexin-6 (SNX6) clone D-5 (Santa Cruz Biotechnology; sc-365965), goat anti-MOMP (Meridian, Memphis, TN), guinea pig anti-L2, sheep anti-IncA (made to order by Seramum Diagnostica GmbH, Heidesee, Germany), rabbit anti-IncE, rabbit anti-IncG, rabbit anti-CT813, and rabbit anti-HctB (kind gifts from T. Hackstadt, NIAID, Rocky Mountain Laboratories, Hamilton, MT), and mouse anti-CT223 (kind gift from R. Suchland, University of Washington, WA, and D. Rockey, Oregon State University, OR). Secondary antibodies used for immunofluorescence were donkey anti-mouse-, rabbit-, or sheep-647, donkey anti-mouse- or rabbit-594, donkey anti-mouse- or rabbit-488, and donkey anti-mouse-, guinea pig-, or rabbit-405 (Jackson ImmunoLabs and Invitrogen). DAPI (4′,6-diamidino-2-phenylindole) was used to visualize DNA as indicated. For Western blots, after incubation with the indicated primary antibody followed by the appropriate secondary antibodies conjugated to IRDye 680LT or IRDye 800 CW (LiCor Biosciences, Lincoln, NE), membranes were imaged using Azure c600 (Azure Biosystems, Dublin, CA) and processed using Adobe Photoshop Creative Cloud (Adobe).

Primary antibodies: anti-α tubulin mouse, 1:50,000 (clone: DM1A, Cat. No. 14450282, eBioscience, San Diego, CA, USA); ALK rabbit mAb, 1:2000 (CST #3633); pALK (Tyr1507) rabbit mAb (CST #14678); Akt mouse mAb, 1:2000 (CST #2920); pAkt (Ser473) rabbit mAb, 1:1000 (CST #9277); Erk 1/2 mouse mAb, 1:1000 (CST #4696); pErk 1/2 (Thr202/Tyr204) rabbit mAb, 1:2000 (CST #4370); STAT3 mouse mAb, 1:1000 (CST #9139); pSTAT3 (Tyr705) mouse mAb, 1:2000 (CST #4113). Secondary antibodies: green-fluorescent goat anti-rabbit IRDye 800CW, 1:10,000 (92632211 LI-COR, Lincoln, NE). Red-fluorescent goat anti-mouse IRDye 680LT, 1:10,000 (92668020 LI-COR, Lincoln, NE).

Protein concentration was measured using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Equal amounts of protein were run on an 8% SDS-PAGE gel. Proteins were transferred to a nitrocellulose membrane (Bio-Rad) and blocked with TBS Odyssey Blocking Buffer (LI-COR Biosciences, diluted 1:1 in TBS prior to use). Primary antibodies directed against active β-catenin (Cat# 8814S, Cell Signaling, 1:1000), total β-catenin (Cat# 610153, BD Biosciences, 1:2000) and α-Tubulin (Cat# T9026, Sigma-Aldrich, 1:500) were diluted in blocking buffer supplemented with 0.1% Tween-20 (TBS-T). Staining was performed overnight at 4°C. Membranes were washed in TBS-T followed by incubation with secondary antibodies (IRDye 680LT (Cat# 926–68021) or IRDye 800CW (Cat# 926–32212) (LI-COR), 1:20000 in TBS-T) for 2 hours. Membranes were washed in TBS-T and incubated in TBS prior to scanning at 700 nm and 800 nm using an Odyssey Fc (LI-COR Biosciences). Image Studio^TM Lite 4.0 software (LI-COR Biosciences) was used to quantify relative protein levels. Background correction was performed according to the manufacturer’s instructions (median of pixels, top/bottom border width of 3).

Osteosarcoma and osteoblast cells were lysed with RIPA Lysis Buffer (Upstate Biotechnology, Charlottesville, VA) plus complete protease inhibitor cocktail tablets (Roche Applied Science, IN). Protein concentrations were calculated by the Bio-Rad Protein Assay (Bio-Rad, CA). Antibodies against PD-L1 (1:1000 dilution), Cas9 (1:1000 dilution) were purchased from Abcam, and β-actin (1:2000 dilution) were purchased from Sigma-Aldrich, respectively. Secondary antibodies IRDye^® 800CW and IRDye^® 680LT were obtained from LI-COR (Biosciences, NE). Western blot analysis was performed as previously reported [35 (link)]. Western blot detection and quantitation was performed by the Odyssey infrared imaging system (LI-COR Biosciences, NE).

To analyze the protein levels, the border and infarct zones of the infarcted hearts or the cells were collected and quickly frozen in liquid nitrogen as reported previously.^[4, ¹³, ^72] To extract the proteins, samples were homogenized with a homogenizer in RIPA lysis buffer containing 20 mm Tris‐HCl (pH 7.4), 1% Triton X‐100, 1% deoxycholate, 10% glycerinum, 150 mm NaCl, 2.5 mm EDTA, and 1 mg mL⁻¹ protease inhibitor cocktail on ice. The samples were placed onto the ice for 30 min and then centrifuged at 12 000 × g for 20 min. The supernatant was collected, and the concentration of proteins was analyzed with a protein quantitation kit (Thermo Fisher, 23223). The loading buffer was added to the proteins, and the samples were boiled at 96 °C for 10 min. For immunoblotting analysis, the proteins (≈30–50 µg) were loaded into the PAGE‐SDS gels. The proteins were separated and transferred to the nitrocellulose membrane and incubated with specific antibodies against IFN‐β (Abclonal, A1575), IFNAR1 (Sino biotechnology, 50469‐RP01), IFNAR2 (Abclonal, A1769), and GAPDH (Proteintech, HRP‐60004) overnight at 4 °C. IRDye 680LT or IRDye 800LT fluorescent secondary antibodies (Li‐COR Bioscience, 926–32212) were used for detection. The Odyssey Infrared Imager (Li‐COR Biosciences) was used to visualize the bands, and the integrated density of the gray value was measured by ImageJ.