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. 2016 Nov 14;7:427. doi: 10.3389/fphar.2016.00427

FIGURE 1.

FIGURE 1

Immunoblots of the expression of UGT2B3, UGT2B3(S316N), and CYP3A1 in Sf-9 cells. To obtain microsomes simultaneously expressing CYP3A1 and UGT2B3/UGT2B3(S316N), Sf-9 cells were transfected with recombinant baculovirus for CYP3A1 and UGT2B3/UGT2B3(S316N). The lanes labeled “UGT2B3” and “UGT2B3(S316N)” show microsomal samples of single transfected cells. The lanes labeled “UGT2B3+CYP3A1” and “UGT2B3(S316N)+CYP3A1” show double transfected cells. Baculosomes (5 μg protein) from UGT2B3 single-expressing Sf-9 cells were electrophoresed (SDS-PAGE). For baculosomes of CYP3A1-UGT2B3 co-expression systems, protein amounts equivalent to UGT2B3 for the single-expressing system were used. Similarly, baculosomes (3 μg protein) from UGT2B3(S316N) single-expressing Sf-9 cells were electrophoresed. For baculosomes of CYP3A1-UGT2B3(S316N) co-expression systems, protein amounts equivalent to UGT2B3(S316N) for the single-expressing system were used. Mock represents the baculosomes (10 μg protein) from Sf-9 cells infected with baculovirus without passenger DNA which served as controls. The proteins in the gel were electrically transferred to a polyvinilidene difluoride membrane, and blotted with goat anti-mouse low pI form UGT (A-C) and rabbit anti-CYP3A2 (D,E), antibodies, respectively. In right panel of (A), baculosomes (25 μg protein) from UGT2B3(S316N)-expressing Sf-9 cells were treated with Endoglycosidase H (EndoH, 500 U) at 37°C for 17 h. Four microgram protein of the digest was subjected to immunoblotting with goat anti-mouse low pI form UGT antibody as a primary antibody. Black and red arrow heads represent original and deglycosylated UGT2B3(S316N), respectively.