Figure 3: NR4A1 interacts and promotes AXIN2–RNF12/ARKADIA complex-mediated SMAD7 degradation.
From: Nuclear receptor NR4A1 promotes breast cancer invasion and metastasis by activating TGF-β signalling
(a,b) Immunoblot analysis of biotinylated TβRI in MDA-MB-231 cells infected with lentivirus expressing (a) or knocking down (b) NR4A1 and treated with TGF-β (5 ng ml−1) for the indicated time points. Co.vec, control empty vector. Co.shRNA, non-targeting shRNA (n=3). Band intensity was normalized to the t=0 controls. Results are shown as means±s.d. of three independent sets of experiments. (c) Immunoblot analysis of immunoprecipitation (IP) samples from HEK293T cells transfected with Flag-SMADs (1–7) and NR4A1 plasmids as indicated. (d) Immunoblot analysis of TβRI and SMAD7 in PC3 cells stably expressing NR4A1 or in which NR4A1 is depleted. SMAD1 immunoblot was taken along as a loading control. (e) Immunoblot (IB) analysis of biotinylated TβRI in HeLa cells transfected with V5-SMAD7 and NR4A1. (f) Immunoblot analysis of SMAD7 expression in NR4A1+/+ and NR4A1−/− MEFs treated with cycloheximide (CHX, 20 μg ml−1) for the indicated time points (n=3). (g) Restoration of the expression of NR4A1 in NR4A1−/− MEFs re-established the faster degradation rates of SMAD7 as observed in wild-type MEFs (n=3). For f and g, Band intensity was normalized to the t=0 controls. Results are shown as means ±s.d. of three independent sets of experiments. (h) HEK293T cells transfected with or without NR4A1 were treated with MG132 (5 μM) overnight and lysed for IP with NR4A1 antibody followed by immunoblot analysis. (i) HEK293T cells infected with lentivirus expressing NR4A1, AXIN2, ARKADIA and RNF12 were treated with MG132 (5 μM) overnight and lysed for the ubiquitination assay followed by immunoblot analysis. (j) HEK293T cells infected with lentivirus expressing NR4A1, RNF12 targeting shRNA (shRNF12) and/or ARKADIA targeting shRNA (shARKADIA) were treated with MG132 (5 μM) overnight and lysed for the ubiquitination assay followed by immunoblot analysis. (k) NR4A1+/+ and NR4A1−/− primary MEF cells infected with Flag-NR4A1 or HA-AXIN2 lentivirus were treated with MG132 (5 μM) overnight and lysed for ubiquitination assay followed by immunoblot analysis. Ub, ubiquitin.
