Phosphatase inhibitor cocktail

Following the different treatments, the cells were harvested and lysed in radioimmunoprecipitation assay buffer (cat no. KGP702; KeyGen Biotech Co., Ltd.) supplemented with 1 mM phenylmethylsulfonyl fluoride (cat no. KGP610; KeyGen Biotech Co., Ltd.) and 1 mM phosphatase inhibitor cocktail (cat no. KGP602; KeyGen Biotech Co., Ltd.). The mixture was centrifuged at 12,000 × g for 20 min and the supernatant was collected. The protein concentration was determined using a BCA assay kit (cat no. KGPBCA; KeyGen Biotech Co., Ltd.) and each sample contained 30 μg protein per 10 μl. The protein samples were mixed with loading buffer (cat no. KGP101; KeyGen Biotech Co., Ltd.) and the proteins were separated using 6 or 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA, USA). Following soaking in a blocking buffer (TBS with 5% nonfat dry milk) for 2 h, the blots were incubated at 4°C overnight with the primary antibody and subsequently incubated at 37°C for 1 h with the horseradish peroxidase-conjugated secondary antibody. The bands were visualized using chemiluminescence and images were captured using a ChemiDoc XRS imaging system (Bio-Rad). These were analyzed using Image Lab software (Bio-Rad).

Retinal homogenates were prepared in ice–cold lysis buffer supplemented with PMSF, protease inhibitor cocktail, and phosphatase inhibitor cocktail (all from KeyGen BioTECH, Nanjing, China) with a tissue homogenizer and cleared via centrifugation (12,000× g for 10 min at 4 °C). The retinal protein concentration was determined using bicinchoninic acid (BCA) kit (KeyGen BioTECH), and equal amounts of total protein were electrophoresed on 10% polyacrylamide gel and subsequently transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA) following a standard protocol. After blocking for 10 min with QuickBlock™ Blocking Buffer (Genscript, Nanjing, China), the membranes were incubated with primary antibodies for 4 °C overnight followed by incubation with HRP–conjugated secondary antibodies for 2 h at room temperature. After washing three times with 0.1% Tween–20/TBS, the immunoreactive proteins were visualized with an enhanced chemiluminescence kit (Millipore) and a chemiluminescence imager (Tanon Science & Technology, Shanghai, China). For stripping, the membranes were incubated in a mild stripping buffer (Epizyme Biomedical Technology, Shanghai, China) for 10 min when necessary. The recorded data and densitometric analysis were quantified using ImageJ. The relative expression of a specific protein was calculated with respect to β–Actin.

Cells were all harvested with cell scrapers after washing 3 times with pre-cold PBS. Then, the harvested cells were centrifuged for 3 minutes at 1000x g, and the pellets were saved. The cell pellets were lysed with RIPA lysis buffer (KeyGen Biotech, Shanghai, China) containing 1 mmol/l phenylmethylsulphonyl fluoride (KeyGen Biotech, Shanghai, China) and 1 mmol/l phosphatase inhibitor cocktail (KeyGen Biotech, Shanghai, China). The mixture was centrifuged for 10 minutes at 12000x g, and the supernatant containing proteins was saved.

Cells were lysed in cold RIPA buffer that had been freshly supplemented with 1 mmol/l phenylmethylsulfonyl fluoride (PMSF), a phosphatase inhibitor cocktail (KeyGEN Biotech, Nanjing, China) and a protease inhibitor cocktail (Amresco, Houston, TX, USA). Total protein concentrations were measured with the BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA). Then, the proteins were separated on 10% SDS-PAGE gels and transferred to nitrocellulose (NC) membranes. Membranes were blocked with 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween 20, incubated with the appropriate primary antibodies, and then incubated with the appropriate HRP-conjugated secondary antibodies. Peroxidase activity was detected with Immobilon Western Chemiluminescence HRP Substrate (Millipore, Billerica, MA, USA). β-actin served as a loading control.

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.