Anti phospho sting ser365

Colon tissues, cells, and EVs fractions were homogenized using the RIPA buffer (Jiangsu KeyGEN BioTECH, Nanjing, China), phosphatase inhibitor cocktail, and protease inhibitor cocktail (Jiangsu KeyGEN BioTECH, Nanjing, China). Pierce BCA Protein Assay Kit (Jiangsu KeyGEN BioTECH, Nanjing, China) was used to determine the protein concentration. Anti-CD63 (A5271, ABclonal), anti-Alix (12422-1-AP, Proteintech), anti-CD81 (66866-1-Ig, Proteintech), anti-Calnexin (ab22595, Abcam), anti-STING (19851-1-AP, Proteintech), anti-Phospho-STING (Ser366) (85735, Cell Signaling Technology), anti-Phospho-STING (Ser365) (72971, Cell Signaling Technology), anti-IRF3 (4302, Cell Signaling Technology), anti-Phospho-IRF3 (Ser396) (29047, Cell Signaling Technology), anti-NF-kB p65 (A19653, ABclonal), anti-Phospho-NF-kB p65 (Ser536) (3303, Cell Signaling Technology), anti-Beta-Actin (4970, Cell Signaling Technology), anti-GAPDH (AF1186, Beyotime Biotechnology) and secondary antibodies (KGP1201, Jiangsu KeyGEN BioTECH, Nanjing, China) were used for western blot.

Immunoblotting was performed as described previously^{39 (link)}. Briefly, cytoplasmic extracts and nuclear extracts were prepared using NE-PER Nuclear and Cytoplasmic Extraction Reagent (Thermo Fisher Scientific). The antibodies used for the immunoblot analysis were anti-SCD2 (Santa cruz), anti-cGAS (Cell signaling), anti-MAVS (Cell signaling), anti–STING (Cell signaling), anti–phosphoSTING (Ser365) (Cell signaling), anti–TBK1 (Cell signaling), anti–phosphoTBK1 (S172) (Cell signaling) and anti-Tublin (Thermo).