Figure 4.
ERAdP interacts with NLRP6. (A) Immunoprecipitation assay was performed using small intestinal crypts from WT and ERAdP-HA mice. Crypts were lysed and incubated with anti-HA antibody and protein A/G beads or only protein A/G beads as control. Proteins precipitated on the beads were resolved by SDS-PAGE, followed by silver staining, and differential bands were cut for mass spectrometry. The band of NLRP6 was shown. (B) co-IP analysis of NLRP6-Myc and ERAdP-Flag. NLRP6-Myc and ERAdP-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) Endogenous co-IP of NLRP6 and ERAdP-HA. Small intestinal crypts from WT and ERAdP-HA mice were lysed and incubated with anti-HA antibody and protein A/G beads. Proteins precipitated on the beads were analyzed with anti-NLRP6 and anti-HA antibodies. β-Actin was used as a loading control. (D) Colocalization analysis of ERAdP-EGFP and NLRP6-mCherry. ERAdP-EGFP and NLRP6-mCherry were cotransfected into Caco-2 cell line, and colocalization was analyzed with fluorescence imaging. Scale bar: 7.5 μm. (E and F) Domain mapping analysis of NLRP6-binding domains of ERAdP protein. Schematic diagram showing truncation mutants of ERAdP (E). FL, full-length; N: N-terminal; TM1, transmembrane 1, TM2, transmembrane 2; C, C-terminal. NLRP6-Myc were transfected into HEK293T cells for 48 h. Cell lysates were incubated with different truncation mutants of GST-ERAdP recombinant protein and GST beads. Proteins precipitated on the beads were analyzed with anti-Myc and anti-GST antibodies. β-Actin was used as a loading control (F). (G) Colocalization analysis of ΔN-ERAdP-EGFP and NLRP6-mCherry. Truncation mutant forms of ΔN-ERAdP-EGFP and NLRP6-mCherry were cotransfected into Caco-2 cell line, and colocalization was analyzed with fluorescence imaging. Scale bar: 3.5 μm. (H) Left: Immunofluorescence staining of SIs from mice administered with DMSO or Ac-YVAD-CMK. Representative Paneth cells and goblet cells are shown. Green: UEA-1; blue: DAPI, red: EpCAM. Scale bar: 20 μm. Right: Diameter of WGA+ cells (n = 10 mice and 20 villus-crypts were analyzed per mouse) and DCVs per Paneth cells were quantified (n = 6 mice and 20 villus-crypts were analyzed per mouse). (I) ELISA of caspase-1 activity examination of crypts from WT and Cnep1r1ΔIEC mice (n = 6 mice for each group). (J) NLRP6 expression levels of SI crypts from littermate WT and Cnep1r1ΔIEC mice were detected by western blot with anti-NLRP6 antibody. β-Actin was used as a loading control. (K) Immunofluorescence staining of ileum crypts from littermate WT and Cnep1r1ΔIEC mice. Red: NLRP6, green: UEA-1, blue: DAPI, and gray: EpCAM. Scale bar: 5 μm. (L) Immunohistochemistry staining of lysozyme in intestines from WT and Nlrp6−/− mice. Scale bar: 35 μm. (M) Left: Electron micrograph of small intestinal crypts in littermate WT and Nlrp6−/− mice. Paneth cells were drawn with dashed white lines, normal DCVs were indicated by yellow asterisk, and abnormal DCVs were indicated by red asterisk. Scale bar: 5 μm. Right: Number of DCVs was calculated (n = 10 mice for each group). Data in H, I, and M are expressed as means ± SEM, then significance was determined by unpaired two-tailed Student’s t test (***P < 0.00; ns, not significant). Data in A–D and F–M are representative of at least three independent experiments. Source data are available for this figure: SourceData F4.

ERAdP interacts with NLRP6. (A) Immunoprecipitation assay was performed using small intestinal crypts from WT and ERAdP-HA mice. Crypts were lysed and incubated with anti-HA antibody and protein A/G beads or only protein A/G beads as control. Proteins precipitated on the beads were resolved by SDS-PAGE, followed by silver staining, and differential bands were cut for mass spectrometry. The band of NLRP6 was shown. (B) co-IP analysis of NLRP6-Myc and ERAdP-Flag. NLRP6-Myc and ERAdP-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) Endogenous co-IP of NLRP6 and ERAdP-HA. Small intestinal crypts from WT and ERAdP-HA mice were lysed and incubated with anti-HA antibody and protein A/G beads. Proteins precipitated on the beads were analyzed with anti-NLRP6 and anti-HA antibodies. β-Actin was used as a loading control. (D) Colocalization analysis of ERAdP-EGFP and NLRP6-mCherry. ERAdP-EGFP and NLRP6-mCherry were cotransfected into Caco-2 cell line, and colocalization was analyzed with fluorescence imaging. Scale bar: 7.5 μm. (E and F) Domain mapping analysis of NLRP6-binding domains of ERAdP protein. Schematic diagram showing truncation mutants of ERAdP (E). FL, full-length; N: N-terminal; TM1, transmembrane 1, TM2, transmembrane 2; C, C-terminal. NLRP6-Myc were transfected into HEK293T cells for 48 h. Cell lysates were incubated with different truncation mutants of GST-ERAdP recombinant protein and GST beads. Proteins precipitated on the beads were analyzed with anti-Myc and anti-GST antibodies. β-Actin was used as a loading control (F). (G) Colocalization analysis of ΔN-ERAdP-EGFP and NLRP6-mCherry. Truncation mutant forms of ΔN-ERAdP-EGFP and NLRP6-mCherry were cotransfected into Caco-2 cell line, and colocalization was analyzed with fluorescence imaging. Scale bar: 3.5 μm. (H) Left: Immunofluorescence staining of SIs from mice administered with DMSO or Ac-YVAD-CMK. Representative Paneth cells and goblet cells are shown. Green: UEA-1; blue: DAPI, red: EpCAM. Scale bar: 20 μm. Right: Diameter of WGA+ cells (n = 10 mice and 20 villus-crypts were analyzed per mouse) and DCVs per Paneth cells were quantified (n = 6 mice and 20 villus-crypts were analyzed per mouse). (I) ELISA of caspase-1 activity examination of crypts from WT and Cnep1r1ΔIEC mice (n = 6 mice for each group). (J) NLRP6 expression levels of SI crypts from littermate WT and Cnep1r1ΔIEC mice were detected by western blot with anti-NLRP6 antibody. β-Actin was used as a loading control. (K) Immunofluorescence staining of ileum crypts from littermate WT and Cnep1r1ΔIEC mice. Red: NLRP6, green: UEA-1, blue: DAPI, and gray: EpCAM. Scale bar: 5 μm. (L) Immunohistochemistry staining of lysozyme in intestines from WT and Nlrp6−/− mice. Scale bar: 35 μm. (M) Left: Electron micrograph of small intestinal crypts in littermate WT and Nlrp6−/− mice. Paneth cells were drawn with dashed white lines, normal DCVs were indicated by yellow asterisk, and abnormal DCVs were indicated by red asterisk. Scale bar: 5 μm. Right: Number of DCVs was calculated (n = 10 mice for each group). Data in H, I, and M are expressed as means ± SEM, then significance was determined by unpaired two-tailed Student’s t test (***P < 0.00; ns, not significant). Data in A–D and F–M are representative of at least three independent experiments. Source data are available for this figure: SourceData F4.

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