Figure 5. ENC1 down-regulated Nrf2 at the translational level.

A. Pulse-chase assay was conducted with MDA-MB-231 cells transfected with either an empty vector or an expression vector for ENC1-Myc. Cells were incubated for 30 minutes with medium containing [³⁵S]-methionine and [³⁵S]-cysteine to label proteins. Cells were then washed and incubated in normal complete medium for the indicated time periods prior to cell lysis. Cell lysates were subjected to immunoprecipitation with anti-Nrf2 antibodies and immunoprecipitates were resolved in SDS-PAGE gel and detected by autoradiography. B. Nrf2 band intensities were quantified using Quantity One (BIO-RAD) and the half-life was plotted and calculated. C. Pulse-chase assay was conducted in the same way, except that cells were treated with 100 µM tBHQ for 4 hr. D. Quantified data of Fig. 5C.