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. 2020 Feb 5;24(5):2802–2818. doi: 10.1111/jcmm.14708

Figure 5.

Figure 5

Foxf2 and Smad6 co‐regulate COL5A2 transcription in the pathogenesis of HESC fibrosis. (A) Co‐IP analysis of the interaction between Foxf2 and Smad6. Cell lysates were immunoprecipitated with anti‐Foxf2 antibody and anti‐Smad6 antibody, respectively, and were subjected to subsequent immunoblotting with anti‐Smad6 or anti‐FoxfF2 antibody. The result confirmed that Foxf2 interacts with Smad6. (B) ChIP analysis of the potential binding sites at the promoter region of COL5A2 that Smad6 bond. DNA fragments immunoprecipitated by anti‐Smad6 antibody were quantified by qPCR using primers covering predicted binding site. The result showed that Smad6 may bind at the promoter region of COL5A2. (C) ChIP analysis of the potential binding sites at the promoter region of COL5A2 that Foxf2 bond. DNA fragments immunoprecipitated by anti‐Foxf2 antibody were quantified by qPCR using primers covering predicted binding site. The result showed that Foxf2 may bind at the promoter region of COL5A2. (D) LV‐Foxf2 was cotransfected with PGL3, PGL3‐COL5A2‐1, PGL3‐COL5A2‐2, PGL3‐COL5A2‐3 into 293T cells (n = 3). Dual‐luciferase assay was performed to detect the activity and showed that Foxf2 bond PGL3‐COL5A2‐3. (E) pcDNA3.1‐Smad6 was cotransfected with PGL3, PGL3‐COL5A2‐1, PGL3‐COL5A2‐2, PGL3‐COL5A2‐3 into 293T cells. Dual‐luciferase assay was performed to detect the activity and showed that Smad6 bond PGL3‐COL5A2‐3. (F) RT‐PCR analysis of the effect of Foxf2 and Smad6 on COL5A2 expression. The result showed Foxf2 promoted COL5A2 expression, but Smad6 inhibited Foxf2‐induced COL5A2 expression