Figure 4. Effect of GA on the distribution of signaling molecules and β₂AR/G protein coupling in plasma membrane.

(A) Lipid raft fractions were purified following a sodium carbonate detergent-free method on a sucrose gradient. The presence of β₂AR, Gα_s, flotillin-1, and caveolin-1 was detected in each fraction by Western blot analysis using the appropriate antibodies. (B) Co-immunoprecipitation of Gα_s and clathrin heavy chains with HA-β₂AR. GA decreased β₂AR binding to clathrin but increased β₂AR binding to Gα_s. Cells were pretreated with 0.1 mM GA, followed by 0.1 mM isoproterenol stimulation for the indicated time. An anti-HA affinity matrix was used to precipitate HA-β₂AR. Co-immunoprecipitated endogenous Gα_s and clathrin heavy chain were detected by Western blot analysis.