Classic magnetic protein a g ip co ip kit

The MRN complex and cGAS co-IP assays were carried out according to the manufacturer’s instructions (Classic Magnetic Protein A/G IP/Co-IP Kit; Epizyme, Shanghai, China). To summarize, specific antibodies (anti-cGAS, Santa Cruz, dilution:1:50 and anti-NBN, Proteintech, Rosemont, IL, USA, dilution: 4 µg/mL) were mixed with protein A/G Magnetic Beads and placed on the rotator overnight at 4°C to generate antibody-magnetic bead complexes; IgG was used as a negative control. Proteins were extracted from AGS cells in 60 mm dishes and lysed using mild lysis for 30 min, then incubated with antibody-magnetic beads complexes overnight at 4°C to form protein-antibody-magnetic beads complexes; the immunoprecipitation complexes were washed and used for further immunoblotting (IB) analysis.

Physical interactions between proteins and the phosphorylation status of target proteins were analyzed by the Classic Magnetic Protein A/G IP/Co-IP Kit (YJ201, Epizyme, China) in conjunction with WB analysis. The inputs used for WB in co-IP assays were 10% whole-cell lysates. For the negative control, bait proteins were captured with Normal Rabbit IgG (2729, Cell Signalling Technology, USA) or Mouse Control IgG (AC011, ABclonal, China). Information on the primary antibodies used to capture bait proteins for the co-IP assays is available in Supplementary file 1—table 1.