Af8382

Total protein was extracted by cell lysis with RIPA buffer and quantified with BCA assay. Next, equal amounts of protein (20µg) were separated via SDS-PAGE, and a PVDF membrane was used for protein transfer. The membrane was incubated with primary antibodies (4°C, overnight) against SLC15A4 (1:1000, ab64429, Abcam, Cambridge, MA), TASL (1:1000, ab69152, Abcam), IRF5 (1:1000, ab181553, Abcam), phospho-IRF5 (1:2000, AF8382, Affinity Biosciences, USA) and GAPDH (1:5000, bs1009R, Bioss, Bejing, China). Then, the membrane was washed with TBST three times (10 min each) and subsequently incubated for 1h with an anti-rabbit IgG HRP-conjugated secondary antibody. The greyscale value of the protein bands was determined with ImageJ software.

CAL-1 cells were transfected with negative controls, miR-31-5p mimics or miR-31-5p inhibitor for 24h and stimulated with R848 for 4h. Then, the cells were washed with PBS and fixed with cold 100% methanol for 10 min, blocked with a BSA/PBS solution, washed with PBS, stained with an anti-phospho-IRF5 antibody (1:200, AF8382, Affinity Biosciences) overnight at 4°C, and incubated with an Alexa Fluor 488 goat anti-rabbit IgG secondary antibody (1:400, ab150077, Abcam, Shanghai, China). The samples were mounted with DAPI containing diamond Prolong mounting solution (AR1176, Boster, Wuhan, China) and were analyzed on a Leica confocal microscope.