Figure 1.
Three common plasticizers (bisphenol-A (BPA), benzyl butyl phthalate (BBP), and di-n-butyl phthalate (DBP)) dose-dependently decreased COMT expression via estrogen receptor (ER) in MCF-7 cells.
Cells were treated with graded doses of each of the three compounds, in the absence or presence of ICI 182,780 (ER antagonist). The resultant changes of soluble-COMT (S-COMT; 23kDa) protein expression were determined by Western blotting. Equal loading of samples were normalized by actin (42kDa). Results are mean ± SEM of four separate experiments (n=4). Statistical significance at the level of *p<0.05 or **p<0.01, as compared to untreated controls. ## p<0.01, as compared between the two designated treatment groups.
Figure 2.
Bacterial expression, purification, and characterization of recombinant MB-COMT protein standard.
(A) Structure of the engineered gene to yield full-length human recombinant membrane-bound-COMT (MB-COMT) protein, and the purity of the recombinant protein by SDS-PAGE following extraction from E. Coli. NE is a novel 18-amino-acid protein tag attached to MB-COMT for detection in the competitive COMT ELISA. (B) Results of SDS-PAGE/Western blotting probed with anti-COMT (lanes 1-4) or anti-NE (lanes 5-8) following immunoprecipitation by either anti-COMT (lanes 1, 2 & 5, 6) or anti-NE (lanes 3, 4 & 7, 8).
Figure 3.
Characterization and application of COMT ELISA.
(A) EC50 determination from mean competitive curve ± S.E.M. (B) Combination plots of 13 individual standard curves illustrating the consistency of the method. (C) Calibration curve used in the ELISA generated from a serial dilution of untreated MCF-7 lysate (COMT concentration: 36.4µg/mg total protein). The lysate gave a linear response under the conditions of the ELISA in a highly similar fashion to that of the purified recombinant MB-COMT standards. (D) Effects of graded doses of 17β-estradiol (E2) on COMT expression in MCF-7 cells determined by COMT ELISA after 48-hour treatment. (E) Effects of treatment of MCF-7 cells with graded doses of BPA, BBP, and DBP, in the presence or absence of ICI 182,780 on COMT expression determined by COMT ELISA. Results are mean ± SEM of four separate experiments (n=4). Statistical significance at the level of **p<0.01, as compared to untreated controls. ## represents significance at level of p<0.01, # p<0.05, between the two designated treatment groups.