Author response image 2. Endogenous human Keap1/Nrf2 expression does not interfere with AR measurement in cell-based assays.

(A) Left, Expression of ectopic Keap1 (labeled here as ‘HTK’, designating the construct used: Halo-TeV-Keap1) is large relative to endogenous hKEAP1 present in cells. HEK293T cells were transfected as indicated in the dual luciferase assay described in Figure 7C-F (original manuscript) and after 42 hours harvested for western blotting. Right, quantification of endogenous (blue bars) and overexpressed Keap1 (red bars) for each transfection condition from n=6 replicates. The affinity of the antibody to each Keap1 variant (Supp. Figure 1D, original manuscript) was used to normalize the Keap1 signal for zKeap1a and zKeap1b. The horizontal dotted line represents the average level of overexpressed hKEAP1. (B) Left, introduction of ectopic Nrf2 alone increases the AR signal by 1000-fold (note: maximal AR is normalized to 100; lowest AR is 0.1). Center and Right; using the dual luciferase assay described in Figure 7C-F (original manuscript), two different siRNAs targeting endogenous hKEAP1 were used to reduce endogenous hKEAP1 levels, while the cells were also transfected to express hNrf2 (Graph at center) only, or low levels of zKeap1a/b (Graph on right), using transfection conditions as in Figure 7D,F (original manuscript). siRNAs have little effect on AR output in either condition. si(control) i, ii, iii correspond to Santa Cruz siRNA controls “A, C, and E (vendor’s labels)”, respectively. si(hKEAP1) i and ii correspond to Dharmacon siGENOME human KEAP1 siRNAs D-012453-03 and D-012453-04. Dharmafect Duo was used for transfection. (C) Left, representative blot for siRNA knockdown efficiency assessment under assay conditions deployed in the original manuscript (data shown is from a single continuous blot); Right, quantification (n=4). Horizontal dotted line represents average level of Keap1 signal of all siControls.