Figure 7.

Detection of disulfide cross-linked mutant M3R homodimers using urea-stripped membranes. Membranes prepared from COS-7 cells transfected with vector DNA or the indicated mutant M3R constructs were subjected to urea stripping (treatment with 7 M urea) prior to incubation with 100 μM Cu-Phen. Membrane extracts were then subjected to SDS-PAGE under nonreducing conditions. Receptor proteins were detected via Western blotting using the polyclonal anti-M3R antibody. Asterisk and arrow indicate putative receptor monomers and dimers, respectively. For control purposes, blots were also probed with a mouse anti-α 1 sodium potassium ATPase monoclonal antibody (predicted molecular mass: 112 kDa). Blot shown is representative of 2 independent experiments.