Flag tag (flag)

The supernatants of WT and Δstm0347 were collected after centrifugation at 7000× g for 20 min and filtered with a 0.22 μm membrane to further remove the remaining bacteria. Sodium deoxycholate was added to a final concentration of 0.2% (wt/vol) before the secreted proteins were precipitated with 20% (wt/vol) trichloroacetic acid (TCA) overnight at 4 °C. Finally, the protein pellets were collected, washed with ice-cold acetone 3 times, and denatured in SDS–PAGE loading buffer with 100 mM Tris-HCl (pH 8.0) at 95 °C for 10 min. Secreted protein samples were loaded for SDS–PAGE electrophoresis and were either transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA) or stained with Coomassie blue. For immunoblotting, membranes were incubated with antibodies and visualized with ECL Western Blotting Substrate (Tanon, Shanghai, China). Antibodies and dilutions were as follows: FLAG (Zen-bio, Chengdu, China, #T201126-3A6, 1:5000) and FliC (Abcam, Cambridge, UK, #ab93713, 1:5000).

MKN45 and MGC803 cells were fixed in 4% paraformaldehyde solution after being transfected with pCDNA3.1-ADAMTS19-3xFlag using a Lipofectamine 3000 reagent and cultured for 48–72 h. The cell membranes were penetrated with a 0.25% Triton X100 (meilunbio, Dalian, China) solution. After 15 min, 1% BSA was transferred to block the cells at room temperature for 30 min. Primary antibodies (Flag-tag: 390002, ZenBio, Sicuan, China, 1:1000; P65: 10745-1-AP, Proteintech, Wuhan, China, 1:200) were incubated at 4 °C for 12–16 h. Primary antibody binding and nuclei were observed using fluorescence secondary antibodies (anti-rabbit: A11034, Invitrogen, 1:200; anti-mouse IgG: A11003, Invitrogen, 1:200) and DAPI (4’,6-diamidino-2-phenylindole; #C1002, Beyotime, Shanghai, China, 1:1000) staining, respectively. Finally, the transfected MKN45 and MGC803 cells were observed and photographed using a confocal microscope (Leica, Weztlar, Germany).