Fig 2. Nedd4L attenuates TGF-β1-induced pro-fibrotic responses through targeting TβRII in lung fibroblasts.
A. Immunoblotting analysis of TGF-β1 (10 ng/ml, 0–4 hour)-treated Nedd4L-V5-transfected HLF cells. B. Immunoblotting analysis of TGF-β1 (10 ng/ml, 15 minutes)-treated Nedd4L siRNA-transfected HLF cells. C. Immunoblotting analysis of TGF-β1 (10 ng/ml, 0, 24, and 48 hour)-treated Nedd4L-V5-transfected Mrc5 cells. D. Immunoblotting analysis of TGF-β1 (10 ng/ml, 24 hour)-treated Nedd4L siRNA-transfected HLF cells. E. Immunoblotting analysis of Nedd4L-V5-transfected Mrc5 cells. F. Immunoblotting analysis of Nedd4L-V5 and TβRII-V5-transfected Mrc5 cells. G. Immunoblotting analysis of Nedd4L and TβRII-V5 levels in the immunoprecipitated complex pulled down by a V5 antibody. H. Coimmunostaining of Nedd4L-HA and TβRII-V5 in double plasmid-transfected Mrc5 cells. Arrows indicate co-localization. I. Immunoblotting analysis of poly-ubiquitinated TβRII levels in the immunoprecipitated complex pulled down by a TβRII antibody in a denatured condition by an in vivo ubiquitination assay. Immunoblots are representative of three independent experiments.