Supplemental Material

This article contains the following supporting material:

• Supplemental Figure 1 - The M1 muscarinic receptor is absent from rafts or caveolae as indicated by Nycodenz gradients.
  MDCK tet-off cells expressing HA-tagged M1 mAChR-GFP were grown for 3 days in 10-cm dishes. Control cells (uninfected) and cells infected with caveolin 1 or caveolin 2 or co-expressing both caveolins (50 pfu/cell of each construct) were incubated for 18 hrs for expression. The cells were then extracted with TX-100 (see methods) and subjected to an equilibrium flotation assay in Nycodenz, fractionated, and analyzed by Western blot. (A) Extracts were evaluated for the distribution of recombinant caveolin 1 and 2. (B) Extracts were evaluated for the distribution of HA-tagged M1 mAChR-GFP under expression of the caveolins. (C) Extracts were evaluated for the distribution of HA-tagged M1 mAChR-GFP in the presence or absence of carbachol or carbachol and caveolin 2. Fractions 1 to 7 are of buoyant-floating proteins. Lane T represents equal fractions of total protein material collected before flotation. Both caveolin 2 and the M1 mAChR were devoid of flotation. Caveolin 2 floated slightly when co-expressed with caveolin 1. Arrows indicate caveolin 1, dashed arrows indicate caveolin 2 bands and arrowheads indicate M1 mAChR.