BBP activates AhR at the cell membrane, which interacts with G proteins. (A) Huh7 cells were transfected with pEGFP-C1-AhR or pEGFP-C1 as a plasmid control. Cells were stimulated by adding DMSO or BBP (1 μM) and then analyzed by real-time TIRF microscopy. Scale bars: 10 μm. The left panel shows Huh7 cells transfected with the pEGFP-C1 plasmid control treated with DMSO (upper) or BBP (lower). The middle panel shows Huh7 cells transfected with pEGFP-C1-AhR and then treated with DMSO (upper) or BBP (lower). The increased intensity of GFP fluorescence indicates AhR expression at the cell membrane, which was induced by BBP. The right panel shows the GFP intensity analyzed by Axio Vision Rel 4.8 software. (B) Huh7 cells were transfected with pEGFP-C1-AhR and then stimulated with BBP (1 μM) before analyzed by real-time confocal microscopy (upper panel), Scale bars: 10 μm. The GFP intensity was analyzed by FV10-ASW 2.1 software (Olympus) (lower panel). (C) Expression of Gαq/11 and Gβ proteins after BBP treatment for the indicated time was detected by immunoblotting. β-actin was used as an internal control. (D) Interaction of AhR with Gαq/11 at the cell membrane after treatment with BBP (1 μM) was imaged by double immunogold electron microscopy. Black arrows indicate Gαq/11, and the white arrows indicate AhR. The localization of G protein and AhR protein are shown. (upper left panel) shows the untreatment group and (Lower left, Right left panel) indicated BBP treatment groups. Scale bars: 500 nm. CM, cell membrane; N, nucleus. (E) Huh7 cells after 30 minutes of treatment with BBP (1 μM) or DMSO as the control. The interaction of AhR with Gαq/11 and Gβ was detected by immunoprecipitation (IP) followed by immunoblot analysis. The IgG were used cell lysates mixed with control and BBP treatment group. Normal rabbit IgG were used as negative control. (F) Huh7 cells were transfected with two different AhR shRNAs as described the Methods or a control shRNA. After treatment with or without BBP (1 μM), AhR, Gαq/11, and Gβ levels were measured by immunoblotting. β-actin was used as an internal control.