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. 2023 May 18;16(3):411–429. doi: 10.1016/j.jcmgh.2023.05.002

Figure 6.

Figure 6

Nox4 suppresses activation of canonical TGF-β signaling in vivo. (A) Scheme representing the canonical TGF-β signaling pathway. (B) Reverse-transcription quantitative PCR analysis of the expression of indicated TGF-β regulatory genes in the WT or Nox4-/- mouse colon tissue; these mice differ from those used for RNA-seq (n = 3). Black dots represent untreated WT mice, and red dots represent untreated Nox4-/- mice. (C) Active TGF-β level in WT (black) or Nox4-/- (red) colon tissue lysates measured by enzyme-linked immunosorbent assay. (D) Immunoblotting of TGF-β and TGFβR1 protein levels in WT or Nox4-/- colon tissue extracts; β-actin was used as an internal control. The band size was quantified by ImageJ. (E) Immunoblotting of pSmad2/3, Smad2/3, and Smad4 protein levels in cytosolic and nucleic fraction extracts from WT and Nox4-/- colon tissues. Smad2/3 was used as the pSmad2/3 control proteins, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and HDAC were used as the cytosol and nucleus control proteins, respectively. The band size was quantified by ImageJ. Representative immunofluorescence images of WT or Nox4-/- colon sections stained with (F) TGF-β (red) and 4′,6-diamidino-2-phenylindole (DAPI) (blue), or (G) TGF-β (green), CD11c (violet), and DAPI. Data are expressed as means ± SD. ∗∗P < .01, and ∗∗∗∗P < .001 compared with WT. Cy, Cytosel; KO, Knock-out; LP, Lamina propria; Nu, Nucleus; P, phosporelated.