Figure 2.

Induction of ARRDC3 expression in MDA-MB-231 breast carcinoma cell line using pSLIK vector system. A, schematic of the tetracycline-inducible pSLIK system for HA-tagged ARRDC3 expression. rtTA3, reverse tetracycline transactivator; TRE, tetracycline response element; LTR, long terminal repeat. B, cell lysates from MDA-MB-231 parental and HA-ARRDC3 pSLIK–expressing cells were collected after 48 h of incubation with DOX treatment at 0, 1, or 10 μg/ml. Cell lysates were immunoblotted to detect endogenous ARRDC3 or HA-ARRDC3 expression. β-Actin expression was detected as a control. The data shown (mean ± S.D. (error bars), n = 3) were quantified by densitometry and shown as the -fold change in ARRDC3 expression relative to 0 min control following doxycycline incubation. Statistical significance was determined by one-way ANOVA (*, p < 0.05; **, p < 0.01; n = 3). C, expression of PAR1 on the cell surface was determined before and after DOX (1 μg/ml) treatment for 48 h and determined by ELISA. The data (mean ± S.D., n = 3) shown as optical density determined at 405 nm are representative of three independent experiments.