Fig. 4: WFDC3 interacts with and stabilizes ERβ protein by inhibiting the ubiquitin-proteasome pathway.

a Cells co-transfected with pEGFP-C1-WFDC3 and pDsRed-Express-C1-ERβ were observed under a confocal microscope. Colocalization was shown by merge (yellow). Nuclei were stained by Hoechest 33342 (blue). b and c WFDC3 interacts with ERβ in the transfected cells. Cells were transiently transfected with the indicated plasmids, and co-immunoprecipitations were performed using an anti-Flag antibody (b) or anti-Myc antibody (c). d and e His-ERβ directly interacted with GST-WFDC3 but not GST alone by in vitro GST pull-down assay (d) and His-tag pull-down assay (e), respectively. f The CHX chase assays show that WFDC3 increases ERβ stability in CRC cells. g Co-treatment with MG132 and CHX partially minimizes the upregulation of ERβ induced by WFDC3 overexpression. h WFDC3 decreases poly-ubiquitination of ERβ. 293 T cells were transfected with Myc-ERβ plasmid, HA-Ub plasmid, and Flag-WFDC3 or Flag-vector plasmids. Cell lysates were immunoprecipitated with an anti-Myc antibody or control IgG, followed by immunoblotting using anti-ubiquitin (Ub). Data are expressed as mean ± SD of at least three independent experiments. Statistics were performed using unpaired Student’s t-test, ***P < 0.001.