Fig. 1.

Western blot analysis of the expression of CYP26A1 wild-type and CYP26A1 variants in COS-1 cells. A. COS-1 cells were transiently transfected with pcDNA3.1 alone (EV, empty vector), pcDNA3.1-CYP26A1*1( WT)-FLAG, pcDNA3.1-CYP26A1*2 (R173S)-FLAG, pcDNA3.1-CYP26A1*3( F186L)-FLAG, and pcDNA3.1-CYP26A1*4( C358R)-FLAG. After 48 hr transfection, cells were washed with medium and incubated with 100 μM at-RA and 0.2 mM NADPH. After 2 hr at 37°C, COS-1 cells were harvested and extracted as described and subjected to electrophoresis on a 10% SDS-polyacrylamide gel as described in Materials and Methods. Proteins were detected with a monoclonal anti-FLAG M2- horseradish peroxidase antibody as described under Materials and Methods. B. The image density of the immunoreactive bands on Kodak Biomax MR Film was quantitated using a Fluorskan 8900. Values represent the means ± S.E of triplicate transfections. Differing amounts of amino-terminal Met-FLAG-BAP-fusion protein (400-1200 fmol) were used as an internal standard for FLAG quantitation (R²=0.98 in the linear regression analysis).