Anti nf kb p65

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.

Cells were subjected to fixing (4% paraformaldehyde (v/v) and then permeabilization (0.4% Triton X-100) and incubated with anti-IL-27 receptor (BIO-RAD), anti-NF-kB p65 (Cell signaling) or anti-HDAC6 antibody (ABclonal) for 2 h. For detection of IL-27 receptor and NF- kB, anti-rabbit Alexa Fluor 488 secondary antibody (Molecular probes) was added to cells and incubated for 1 h. For detection of HDAC6, anti-goat Alexa Fluor 546 (Molecular probes) was employed. For fluorescence imaging, confocal laser scanning microscope and software (Fluoview version 2.0) with a X 60 objective (Olympus FV300, Tokyo, Japan) were employed. The lists of antibodies are described in Supplementary Table.