Figure 5. SMAD3 K53/K333 methylation is essential for its membrane localization.

(A) Membrane and cytosolic fractions from MDA-MB-231^SMAD3–/– cells stably transfected with WT SMAD3 or SMAD3 K53/333R plasmids were collected and subjected to IB analysis. (B) Membrane fractions from MDA-MB-231^SMAD3–/– cells stably transfected with WT SMAD3 or SMAD3 K53/333R plasmids and treated with TGFB1 (5 ng/mL) were collected and subjected to IB analysis. (C) MDA-MB-231^SMAD3–/– cells were stably transfected with WT SMAD3 or SMAD3 K53/333R plasmids and treated with TGFB1 (5 ng/mL). IF images show the cellular localization of SMAD3. Scale bar: 10 μm. (D) Membrane and cytosolic fractions from MDA-MB-231 cells silenced with control or EZH2 shRNA (no. 1) were collected and subjected to IB analysis. (E) HEK293T cells were transfected with WT HA-SMAD3 or HA-SMAD3 K53/333R plasmids and treated with TGFB1 (5 ng/mL), and WCEs were collected for IP with anti-HA antibody, followed by IB analysis. (F) HEK293T cells were silenced with control or EZH2 shRNA (nos. 1 and 2), and WCEs were collected for IP with anti-SMAD3 antibody, followed by IB analysis. (G) Co-IP of endogenous SMAD3 from MDA-MB-231 cells transfected with EZH2^WT or EZH2^H689A, followed by IB analysis. (H) Co-IP of endogenous SMAD3 from MDA-MB-231 cells transfected with EZH2^WT or EHZ2^Y641H, followed by IB analysis. All immunoblotting was performed 3 times, independently, with similar results.