FIGURE 4. Rat brain SET interacts with the i3 loop of both the M2- and M3-MR.

GST fusion proteins encompassing the i3 loop of the M2- or M3-MRs (GST-M2-i3 His²⁰⁸—Arg³⁸⁷ and GST-M3-i3 Arg²⁵²—Gln⁴⁹⁰) were expressed in BL21 cells and purified as described under “Experimental Procedures.” The purified GST fusion proteins were electrophoresed on denaturing polyacrylamide gels (10%) and visualized by Coomassie Blue staining. The purified GST fusion proteins were incubated with rat brain cytosol, and the bound proteins were eluted and separated on denaturing polyacrylamide gels (10%), and membrane transfers were immuno-blotted with a polyclonal anti-SET antibody.