Identification of ANXA2 and generation of ANXA2-deficient mice. (A) Peptides of ANXA2 were identified by mass spectrometry. (B and C) Domain mapping analysis of ANXA2-binding domains of NLRP6 protein. Schematic diagram showing truncation mutants of NLRP6 (B). Different truncation mutants NLRP6-Myc and ANXA2-HA were transfected into HEK293T cells for 48 h. Cell lysates were incubated anti-Myc beads. Proteins precipitated on the beads were analyzed with anti-Myc and anti-HA antibodies. β-Actin was used as a loading control (C). (D) Relative Anxa2 expression levels of intestinal crypts from WT and DKO mice were detected by qRT-PCR. Fold changes were normalized to endogenous 18S (n = 6 mice for each group). (E) Construction diagram of ANXA2 KO mouse with CRISPR-Cas9. (F) Diagram of mice injected with AAV to KO ANXA2 and then infected with S. Typhimurium. (G) DNA electrophoresis for Cas9 knock-in validation. (H) Western blot for ANXA2 KO validation with ANXA2 antibody. β-Actin was used as a loading control. (I) Immunofluorescence staining of intestinal organoids from littermate sgCtr and sgAnxa2 mice. Green: UEA-1, red: EpCAM, and blue: DAPI. Scale bar: 10 μm. (J) Intestinal lumen (left) and tissue-associated (right) bacterial load analysis, quantified by qPCR of 16S rRNA gene copy number in distal ileums of WT and sgAnxa2 mice (n = 6 mice for each group). (K) qPCR detection of ileal luminal commensal bacteria classified by phylum (n = 6 mice for each group). Data in D, J, and K are shown as means ± SEM; significance was determined by unpaired two-tailed Student’s t test (*P < 0.05; ***P < 0.001; ns, not significant). Data in A, C, D, and G–K are representative of at least three independent experiments. Source data are available for this figure: SourceData FS4.