AhR Antibody - #AF6278

Figure 6 The alleviation of Vag-induced mastitis in mice by 5-HIAA is dependent on AhR (A and B) Vag induced reduction of AhR protein expression and relative intensity in mammary glands (n = 3). (C and D) The antagonist CH223191 effectively suppressed AhR protein expression and relative intensity in the mammary gland (n = 3). (E) Representative H&E images among the different groups indicated that the antagonism of AhR abolished the protective function of 5-HIAA (n = 7; scale bar, 50 μm). (F) Histopathological score of the mammary gland (n = 6). (G–I) CH223191 treatment increased TNF-α (G), IL-1β (H), and MPO activity (I) in comparison to Vag+5-HIAA treatment (n = 7). (J–M) Pretreatment with CH223191 reduced the expression and relative intensity of TJ proteins ZO-1, Occludin, and Claudin-3 (n = 3). (N) The expression location and level of TJ protein were detected by immunohistochemistry (scale bar, 20 μm). (O and P) Pretreatment with CH223191 eliminated the decreasing effect of 5-HIAA on TNF-α and IL-1β mRNA levels in LPS-induced mouse mammary epithelial cells (n = 6). Data are presented as means ± SDs, and one-way ANOVA was performed for statistical analysis. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; and ∗∗∗∗p < 0.0001 indicate significant differences. See also Figure S8.

FIGURE 7 Effects of emodin and AhR inhibitor CH223191 on the protein expression levels of AhR and CYP1A1 in MCF-7 cells (mean ± SD, n = 3) (A) In the cells treated with emodin, the expression of AhR protein using GAPDH as a loading control and the graphical representations of the AhR/GAPDH ratio; (B) In the cells treated with emodin, the expression of CYP1A1 protein using GAPDH as a loading control and the graphical representations of the CYP1A1/GAPDH ratio; (C) In the cells treated with CH223191, the expression of CYP1A1 protein using GAPDH as a loading control and the graphical representations of the CYP1A1/GAPDH ratio; (D) In the cells intervened with emodin and 10 μmol/L of CH223191, the expression of CYP1A1 protein using GAPDH as a loading control and the graphical representations of the CYP1A1/GAPDH ratio.

Fig. 8 Fn induces AHR nuclear translocation and subsequently results in enhanced CYP1A1 expression. (A) Western blotting analysis of the protein level of AHR in cytoplasm and nucleus after treatment with Fn for 0, 12 and 24 h. (B) KYSE-450 cells cocultured with or without Fn for 24 h after being pretreated with AHR antagonist (CH-223191, 10 μm, 2 h). qRT-PCR analysis of AHR and CYP1A1 expression. (C) Protein levels of AHR in nucleus of Fn-infected cells and control cells with or without CH-223191 pretreatment. (D) An immunofluorescence assay measured the distribution of AHR in the cells. Original magnification: 400×; scale bar = 20 μm. (E) Western blotting results showed that CH-223191 downregulated the CYP1A1 expression and p-AKT level. (F, G) A cell counting assay and colony formation assay were performed to evaluate the effect of CH-223191 pretreatment on the cell proliferation of Fn-infected cells. Data are presented as the mean ± SD from three independent experiments (n = 3) (Student's t-test). *P 

FIGURE 6 | Compared with that in the control group, decreased IL-10 and TGF-β expression in colon tissues of mice was induced by DSS and detected by immunohistochemistry.Representative photographs of immunohistochemical staining of IL-10 and TGF-β at the indicated time point in each group (A). Scale bars = 50 µm. (B) The mRNA level of IL-10 and TGF-β in colon tissues of different groups. (C) Protein expression of AhR,IL-10, and TGF-β in colon tissues of different groups.

FIGURE 5 | Compared with that in the control group, decreased AHR expression in colon tissues of mice was induced by DSS and detected by immunohistochemistry (A), Representative colon images at the indicated time point in each group. Scale bars = 50 µm. Representative example of an immunofluorescence double staining of Foxp3 (Red) and AhR (Green) in colon tissues (B).

FIGURE 5 | Compared with that in the control group, decreased AHR expression in colon tissues of mice was induced by DSS and detected by immunohistochemistry (A), Representative colon images at the indicated time point in each group. Scale bars = 50 µm. Representative example of an immunofluorescence double staining of Foxp3 (Red) and AhR (Green) in colon tissues (B).

Figure 4. Changes in aryl hydrocarbon receptor (AHR) and reactive oxygen species (ROS) after 5-HIAA intervention in neutrophils. (A) Immunofluorescence staining of cell crawls of each experimental group, AHR in red, Ly6G-labeled neutrophils in green, and 4′,6-diamidino-2-phenylindole-labeled nuclei in blue. Arrowheads mark AHR nuclear expression in neutrophils secreting NET-like reticulated substances, original magnification: ×630. (B) ROS in the cell culture fluid of each group. Multiple comparisons of groups, *p