Fig. 5: NEDD4-2 is implicated in Wnt/β-catenin/TGF-β signaling in CCD cells.
CRISPR–Cas9 was used to generate CCD-N21 clones lacking NEDD4-2 or wild-type control clones. A Immunoblot analysis of NEDD4-2 and Vimentin with GAPDH as a loading control. B qRT-PCR analysis of Vimentin relative to TBP in untreated cells, 2.5 ng/ml TGF-β1 or 10 mM LiCl treated for 72 h, n = 4 for 4 independent clones each. C Immunoblot analysis of NEDD4-2, vimentin, and β-catenin with GAPDH as a loading control, untreated or after 72 h TGF-β1 stimulation. Numbers indicate quantitation of β-catenin bands relative to untreated wild type lane, normalized to GAPDH. D Immunoblot analysis of β-catenin after separation of cytoplasmic (marked by α-tubulin) and nuclear (marked by Histone 3, H3) fractions. Numbers indicate quantitation of β-catenin bands relative to untreated wild-type lane, normalized to α-tubulin for a cytoplasmic fraction or H3 for nuclear fraction. E Immunostaining of β-catenin (green) with and without TGF-β1 treatment. DNA is stained by Hoechst (blue). Scale bar: 50 μm. F Immunoblot analysis of pSMAD2/3 and total SMAD2/3 with GAPDH as a loading control. Numbers indicate quantitation of pSMAD bands relative to untreated wild-type lane, normalized to β-actin. G qRT-PCR analysis of Vimentin relative to TBP in untreated or 72 h 500 nM LY-364947 treated Nedd4-2 KO cells, n = 4. Data presented as means ± SEM (B, G) and significance determined using unpaired two-tailed Student’s t test. *P < 0.05, **P < 0.01. Immunoblots are representative of three experiments with similar results.
