Figure 5.
The ERAdP–NLRP6 association recruits ANXA2 onto the DCV membrane for generation of DCVs. (A) GST pull-down showing interaction of NLRP6 and ANXA2. Lysed small intestinal crypts from WT mice were incubated with GST-NLRP6 recombinant protein or GST recombinant protein as control. Proteins precipitated on the beads were resolved by SDS-PAGE, followed by silver staining, and differential bands were cut for mass spectrometry. Representative protein ANXA2 is shown. (B) co-IP analysis of NLRP6-Myc and ANXA2-Flag. NLRP6-Myc and ANXA2-Flag were cotransfected into HEK293T cells for 48 h. Cell lysates were incubated with anti-Flag antibody for immunoprecipitation; proteins precipitated on the beads were analyzed by western blotting with anti-Myc and anti-Flag antibodies. β-Actin was used as a loading control. (C) co-IP analysis of NLRP6-Myc and ANXA2-HA under the condition of absence or presence of ERAdP-Flag. β-Actin served as a loading control. (D) Western blot showing ANXA2 expression levels of SI crypts from littermate WT, Cnep1r1ΔIEC, Nlrp6−/−, and ERAdP–NLRP6 DKO mice with anti-ANXA2 antibody. β-Actin was used as a loading control. (E) Immunofluorescence staining showing ANXA2 localization in ileum crypts from littermate WT, Cnep1r1ΔIEC, Nlrp6−/−, and DKO mice. Red: ANXA2, green: UEA-1, blue: DAPI, and gray: EpCAM. Scale bar: 5 μm. (F) Left: Immunofluorescence staining showing DCVs in Paneth cells from littermate WT and sgAnxa2 mice. Green: UEA-1, red: EpCAM, and blue: DAPI. Scale bar: 20 μm. Right: Number of DCVs per Paneth cells was quantified (n = 6 mice for each group). (G) Left: 3D immunofluorescence staining of ileum crypts from WT and sgAnxa2 mice. Green: UEA-1; red: EpCAM; yellow: DCVs’ surface fitted by Imaris 9 software. Scale bar: 20 μm. Right: Volume of DCVs was quantified (n = 6 mice for each group). (H) Left: DIC images of organoids from WT, Cnep1r1ΔIEC, DKO, sgAnxa2, and ANXA2-overexpressing DKO mice. Organoids were stimulated by CCh for 10 min and then washed out. Images were captured before (0 h) and after CCh stimulation (24 h). Areas of DCV were drawn with dashed red lines, and the corresponding Paneth cells were drawn with dashed black lines. Scale bar: 10 μm. Right: Area of DCVs was quantified (n = 6 mice for each group). (I) Lysozyme in supernatant of stimulated crypts from WT, DKO, and sgAnxa2 mice after treatment of DMSO or CCh was measured by ELISA (n = 6 mice for each group). (J) Bacterial load analysis of S. Typhimurium in crypt supernatants. SI crypts of littermate WT, DKO and sgAnxa2 mice were isolated and stimulated by DMSO or CCh. The crypt supernatants were incubated with S. Typhimurium, and CFU was measured (n = 6 mice for each group). (K) Immunofluorescence staining of ileum crypts from mice of control, ABX mock treated, or treated through intragastric gavage with c-di-AMP. Green: UEA-1, blue: DAPI, red: ANXA2, and gray: EpCAM. Scale bar: 10 μm. Data in F–J are shown as means ± SEM, then significance was determined by unpaired two-tailed Student’s t test (**P < 0.01; ***P < 0.001; ns, not significant). Data above are representative of at least three independent experiments. Source data are available for this figure: SourceData F5.

The ERAdP–NLRP6 association recruits ANXA2 onto the DCV membrane for generation of DCVs. (A) GST pull-down showing interaction of NLRP6 and ANXA2. Lysed small intestinal crypts from WT mice were incubated with GST-NLRP6 recombinant protein or GST recombinant protein as control. Proteins precipitated on the beads were resolved by SDS-PAGE, followed by silver staining, and differential bands were cut for mass spectrometry. Representative protein ANXA2 is shown. (B) co-IP analysis of NLRP6-Myc and ANXA2-Flag. NLRP6-Myc and ANXA2-Flag were cotransfected into HEK293T cells for 48 h. Cell lysates were incubated with anti-Flag antibody for immunoprecipitation; proteins precipitated on the beads were analyzed by western blotting with anti-Myc and anti-Flag antibodies. β-Actin was used as a loading control. (C) co-IP analysis of NLRP6-Myc and ANXA2-HA under the condition of absence or presence of ERAdP-Flag. β-Actin served as a loading control. (D) Western blot showing ANXA2 expression levels of SI crypts from littermate WT, Cnep1r1ΔIEC, Nlrp6−/−, and ERAdP–NLRP6 DKO mice with anti-ANXA2 antibody. β-Actin was used as a loading control. (E) Immunofluorescence staining showing ANXA2 localization in ileum crypts from littermate WT, Cnep1r1ΔIEC, Nlrp6−/−, and DKO mice. Red: ANXA2, green: UEA-1, blue: DAPI, and gray: EpCAM. Scale bar: 5 μm. (F) Left: Immunofluorescence staining showing DCVs in Paneth cells from littermate WT and sgAnxa2 mice. Green: UEA-1, red: EpCAM, and blue: DAPI. Scale bar: 20 μm. Right: Number of DCVs per Paneth cells was quantified (n = 6 mice for each group). (G) Left: 3D immunofluorescence staining of ileum crypts from WT and sgAnxa2 mice. Green: UEA-1; red: EpCAM; yellow: DCVs’ surface fitted by Imaris 9 software. Scale bar: 20 μm. Right: Volume of DCVs was quantified (n = 6 mice for each group). (H) Left: DIC images of organoids from WT, Cnep1r1ΔIEC, DKO, sgAnxa2, and ANXA2-overexpressing DKO mice. Organoids were stimulated by CCh for 10 min and then washed out. Images were captured before (0 h) and after CCh stimulation (24 h). Areas of DCV were drawn with dashed red lines, and the corresponding Paneth cells were drawn with dashed black lines. Scale bar: 10 μm. Right: Area of DCVs was quantified (n = 6 mice for each group). (I) Lysozyme in supernatant of stimulated crypts from WT, DKO, and sgAnxa2 mice after treatment of DMSO or CCh was measured by ELISA (n = 6 mice for each group). (J) Bacterial load analysis of S. Typhimurium in crypt supernatants. SI crypts of littermate WT, DKO and sgAnxa2 mice were isolated and stimulated by DMSO or CCh. The crypt supernatants were incubated with S. Typhimurium, and CFU was measured (n = 6 mice for each group). (K) Immunofluorescence staining of ileum crypts from mice of control, ABX mock treated, or treated through intragastric gavage with c-di-AMP. Green: UEA-1, blue: DAPI, red: ANXA2, and gray: EpCAM. Scale bar: 10 μm. Data in F–J are shown as means ± SEM, then significance was determined by unpaired two-tailed Student’s t test (**P < 0.01; ***P < 0.001; ns, not significant). Data above are representative of at least three independent experiments. Source data are available for this figure: SourceData F5.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal