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. 2012 Dec;343(3):712–724. doi: 10.1124/jpet.112.198770

Fig. 3.

Fig. 3.

Effect of increasing UGT2A1_i2 expression on UGT2A1_i1 glucuronidation activity. A, representative Western blots showing stable expression of UGT2A1_i1_V5 and induction of UGT2A1_i2_FLAG expression in HEK293 cells. UGT2A1_i2_FLAG expression was induced through the addition of increasing doses of PonA for 12 h; 50 μg of total protein homogenate from each PonA treatment group was loaded per lane for each Western blot. B, Western blots with 100 μg of protein from the control and 10-μM PonA treatment groups was performed with an anti-UGT2A1 antibody to confirm UGT2A1 expression levels detected by the anti-V5 and anti-FLAG antibodies. For A and B the relative expression of UGT2A1_i1, and the relative UGT2A1_i2/UGT2A1_i1 ratio, was determined for each treatment group. Induction of UGT2A1_i2 expression and Western blots were performed in triplicate. In all cases, β-actin was used as a loading control. C, representative chromatograms showing the effect of increasing UGT2A1_i2 expression on UGT2A1_i1 activity against 1-OH-pyrene. D, Michaelis-Menten kinetic curves for 1-OH-pyrene, 3-OH-B[a]P, and B[a]P-7,8-diol glucuronidation by UGT2A1_i1, showing the effect of increasing expression levels of UGT2A1_i2 on UGT2A1_i1 enzyme activity.