Stag2

Cells were lysed in a modified lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 50 mM NaF, 10 mM β-glycerophosphate, 10 nM calyculin A, 1 mM Na3VO4, and protease inhibitors), followed by SDS-PAGE and western blotting analysis as previously described^{55 (link)}. The primary antibodies against STAG2 (Santa Cruz, SC-81852), IRF9 (Cell Signaling, #76684), IRF7 (Cell Signaling, #4920), USP18 (Cell Signaling, #4813), ISG15 (Santa Cruz, SC-166755), IRF1 (Cell Signaling, #8478), IRF3 (Cell Signaling, #11904), GAPDH (Cell Signaling, #2118), GAPDH (Cell Signaling, #51332), STAG1 (Novus Biologicals, NB100-298), PD-L1 (R&D Systems, AF156), CTCF (Cell Signaling, #2899), and Flag M2 (Sigma, F3165) were used. All primary antibodies were used at a 1:1,000 dilution, with the exception of GAPDH (1:3000) and PD-L1 (1:200).

Total protein was isolated from camel tissues using lysis buffer (Solarbio, Beijing, China) and quantified using a BCA protein assay kit (Boster). Samples equivalent to 50 μg of protein were resolved using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and were electrotransferred to PVDF membranes (Millipore CAT, Billerica, MA, USA). The membranes were blocked for 2 h in Tris–HCl buffer containing 5% (w/v) non-fat milk powder, then were incubated overnight at 4 °C with the following primary antibodies from mice or rabbits: ACTB (1:5000, Proteintech, China), CAMK2G (1:1000, Proteintech), FST (1:1000, Proteintech), NR5A1 (1:500, Proteintech), PAK1 (1:1000, Santa Cruz, USA), PRL (1:1000, Santa Cruz), STAG2 (1:1000, Santa Cruz), TGFB2 (1:500, Santa Cruz), Wnt2b (1:500, Santa Cruz), GAPDH (1:5000, Proteintech). Membranes were then washed three times with PBS containing 0.1% Tween 20, incubated with goat anti-mouse/anti-rabbit IgG conjugated to horseradish peroxidase for 2 h at room temperature (1:5000, Bioss, Beijing, China), then washed three times with PBS containing 0.1% Tween 20. All immunoblot assays were performed at least three times. Optical densities of the bands were quantified, and membranes were scanned using Image-Pro plus 6.0 (Media Cybernetics Co., Rockville, MD, USA).