Co-immunoprecipitation (Co-IP) and immunoblotting (IB) assays

Co-IPs were performed using COS-7 cells or HEK-293T cells growing in 6-cm tissue culture plates. Cells were transfected to express the indicated proteins and treated without or with 1000 U/mL recombinant human IFN-α (PBL Assay Science, Cat#PBL-11200-2) for 0.5 h (analysis of endogenous STAT1) or 1 h (analysis of STAT1-GFP) before lysis using lysis buffer (10 mM Tris/Cl pH 7.5; 150 mM NaCl; 0.5 mM EDTA; 0.5% NP-40, 1 × Protease Inhibitor Cocktail (PIC; Sigma-Aldrich Cat #11697498001) and 1x PhosSTOP) for 30 min at 4°C. Supernatants were collected by centrifugation at 12,000 g for 10 min at 4°C and 10% of the cleared lysate was collected for ‘input’ analysis; the remaining lysate was subjected to IP using 10 μL of GFP-Trap beads (Chromotek). Beads were washed 3 times with dilution buffer (10 mM Tris/Cl pH 7.5; 150 mM NaCl; 0.5 mM EDTA, 1 × PIC, 1x PhosSTOP). Protein for IB analysis was eluted by resuspension of beads in SDS-PAGE sample loading buffer and incubation at 90°C for 10 min. The lysate (input) and IP samples were separated by SDS-PAGE before analysis by western blot using antibodies for GFP (Roche Applied Science, catalog no. 11814460001), mCherry (Abnova, catalog no. {"type":"entrez-protein","attrs":{"text":"PAB18013","term_id":"1236630928","term_text":"PAB18013"}}PAB18013 or Abcam, catalog no. ab167453), FLAG (Sigma-Aldrich, catalog no. F1804), STAT1 (BD Biosciences, catalog no. 610185 or Cell Signaling Technology, catalog no. 14994) or pY-STAT1 (Cell Signaling Technology, catalog no. 9176).