Figure 1. Effect of EEAC on P-gp mRNA and protein expression level.

(a)(b) Western blot of P-gp expression with C219 monoclonal antibody (2 µg protein/lane). The upper one was the mature fully glycosylated (∼170 kD) and the lower one was the immature P-gp (∼150 kD). The expression of β-actin was used as loading control. Treatment of EEAC 5 µg/mL for 48 and 72 hours demonstrated no effect on P-gp protein expression. (c)(d)(e) Analysis of mRNA levels with real-time quantitative RT-PCR. There were no significant ABCB1 mRNA expression differences after treating the P-gp expressed cells with 5 µg/ml EEAC for 24, 48 and 72 hours. (f) Cytotoxicity study of EEAC by MTT assay. The IC₅₀ of EEAC for Flp-In™ 293, ABCB1 1236C-2677G-3435C (wild-type), ABCB1 1236T-2677T-3435T and ABCB1 1236T-2677A-3435T were 21.27, 29.31, 20.60 and 22.74 µg/ml, respectively. There was no significant difference among the IC₅₀. Data were presented as mean ± SE of at least three experiments, each in triplicate.