Smurf2 protein is downregulated in triple-negative breast cancer (TNBC) cell lines without concomitant decreases in Smurf2 mRNA. (A) Immunoblotting for Smurf2 in the following cell lines: MCF-10A, untransformed human mammary epithelial cells; MCF-7 and T47D, mammary carcinomas with expression of the estrogen and progesterone receptors (ER+/PR+); MDA-MB-231, BT549, MDA-MB-436, DU4475 and MDA-MB-468, TNBCs; BT474 and SK-BR-3, mammary carcinomas with HER2 amplification. The bar graph indicates relative levels for Smurf2 protein in the cancer cell lines to that in MCF-10A cells. The density of each Smurf2 signal on immunoblots was normalized by that of α-tubulin. Data are shown as mean + SEM from at least three experiments, and the asterisks indicate statistically significant (p < 0.05) differences from the level in MCF-10A cells. (B) Real time PCR analysis for Smurf2 mRNA. Data are normalized by signals for GAPDH mRNA, and presented as relative expression levels to that in MCF-10A cells. (C) Degradation of Smurf2 protein assessed by treatment with cycloheximide (CHX). MCA-10A, MDA-MB-231 and BT549 cells were treated with 100 μg/ml CHX for the indicated hours, and lysates were analyzed by immunoblotting for Smurf2 and α-tubulin.