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

FIGURE 5.

FIGURE 5

Detection of an interaction between CYP3A1 and UGT2B3/UGT2B3(S316N) by His-tag pull-down assay. UGT2B3-HA and UGT2B3(S316N)-HA were expressed in the presence and absence of coexpressed His-CYP3A1. The pull-down assay was performed according to methods previously published (Miyauchi et al., 2015) with slight modification. Microsomes (2 mg protein/mL) were solubilized with sodium cholate. Then the solubilized microsomes were used in each assay. His-CYP3A1 and proteins trapped by this P450 were eluted with buffer containing imidazole at a high concentration. Each protein was detected by immunoblotting with a specific antibody: rabbit anti-HA or rabbit anti-His. Solubilized microsomes and His-CYP3A1–trapped samples are indicated as Input (30% of the input) and PD (pull down), respectively. In (A), HA-tagged wild-type UGT2B3 (WT-HA) was used. In (B), HA-tagged UGT2B3(S316N) (MT-HA) was used. WT-HA+CYP-His and MT-HA+CYP-His indicate microsomes coexpressing UGT2B3-HA or UGT2B3(S316N)-HA with His-CYP3A1, respectively. Duplicate assays were carried out. For each pull-down sample, one half of the precipitate was subjected to SDS-PAGE for detection with either HA or His while the lanes shown by “C” represent a negative control sample obtained by the same procedures but using solubilized control microsomes as an input, with microsomes prepared from Sf-9 cells transfected with control baculovirus alone. Details are described in the Section “Materials and Methods”.