Figure 6.

Presence of AT1R–CB₁R heteromers in activated HSCs. (A) Expression levels of CB₁R, AT1R, and α-SMA by western blotting analysis in HSCs from control (cHSC) and ethanol (eHSCs) treated rats. Calnexin is used as a loading control. (B) Association of AT1R and CB₁R in eHSCs. Lysates from cHSCs and eHSCs were subjected to immunoprecipitation using an anti-AT1R antibody (1 μg)/protein A/G agarose, and to western blotting analysis with CB₁R antibody (1:500). CB₁R is detected in the AT1R immunoprecipitate from eHSCs (and not cHSCs). (C) Detection of AT1R–CB₁R heteromers with heteromer-selective monoclonal antibodies. Receptor abundance was determined in cHSCs and eHSCs with a monoclonal antibody to AT1R–CB₁R by ELISA. Results represent the means±s.e.m. obtained with antibodies from seven different hybridoma clones. ^***P<0.001. (D) Colocalization of AT1R and CB₁R in activated HSCs. eHSCs grown on coverslips were fixed with 4% PFA, permeabilized with 0.1% Triton X-100, and stained with primary rabbit polyclonal anti-CB₁R (1:500) and goat polyclonal anti-AT1R (1:200) antibodies. After fluorescent secondary antibody staining, the coverslips were mounted with mowiol. Slides were examined with a Leica SP5 confocal microscope.