Fig. 3.

Mutation at Thr⁵⁷ does not affect hPXR protein expression levels. HepG2 (A) and COS7 (B) cells were transfected with either wild-type or mutant FLAG-hPXR plasmids. Twenty-four hours post-transfection, the cells were treated with either 0.1% DMSO (indicated by a “–” symbol) or 10 μM rifampicin (indicated by a“+” symbol). Whole cell lysates were collected 24 h after treatment with DMSO or rifampicin and subjected to Western blotting analysis using anti-FLAG and anti-actin antibodies as described under Materials and Methods. pcDNA3-FLAG vector transfection (lane 1) and no transfection (lane 2) are “negative” controls. Wild-type FLAG-hPXR protein expression was shown in lanes 3 and 4. Protein expression for the alanine mutant (FLAG-hPXR^T57A) was shown in lanes 5 and 6 and for the aspartate (FLAG-hPXR^T57D) in lanes 7 and 8. Actin protein expression was analyzed as a mean of loading control. Data shown are from a representative experiment.