Figure 4

The regulation of SNAI2 by DEAR1. (a) Bar plots of qPCR results (mean ± SD) of SNAI2 mRNA levels in control (CshR), DEAR1-KD (DshR), DEAR1-KD/shRCt clone (DshR/shC), and DEAR1-SMAD3 double KD (DshR-shSM3) across 2 runs. P-values were calculated using Wilcoxon Rank Sum tests. *p < 0.05–0.1, **p < 0.01, ***p < 0.001. The barplots were created using RStudio (Version 1.0.143), https://www.rstudio.com. (b) Western blot showing SNAI2 protein expression in control (CshR) and DEAR1-KD clones (DshR) with and without TGF-β treatment. Full length gels for Western blots are shown in Supplementary Fig. S2. (c) Co-immunoprecipitation (Co-IP) to determine possible interaction between DEAR1 and SNAI2. HEK293T cells were co-transfected with an HA-DEAR1-expressing plasmid and/or a Myc-SNAI2 expression plasmid. After 24 h post-transfection, cell lysates were immunoprecipitated with anti-HA (rabbit) antibody. SNAI2 proteins were blotted with anti-Myc (mouse) antibody. Full length gels for Western blots are shown in Supplementary Fig. S3. (d) Co-IP assay to detect ubiquitination of SNAI2 by DEAR1. HEK293T cells were co-transfected with Myc-SNAI2, HA-ubiquitin, and DEAR1. Cells were lysed 48 h post-transfection. Lysates were denatured and immunoprecipitated with anti-Myc (mouse) antibody. Ubiquitin was blotted with anti-HA (Rat) antibody. Full length gels for Western blots are shown in Supplementary Fig. S4.