Figure 3.

Agonist treatment promotes ubiquitination of β-arrestin 2 in MEF KO1/2 cells expressing the mAChRs and β-arrestin 2. MEF KO1/2 cells transfected with FLAG-β-arrestin 2 and either eGFP-M₁ or HA-M₂ mAChR. 24 hr following transfection, cells were treated with 1 mM carbachol for the indicated times. Top panel: cells were lysed and immunoprecipitated (IP) with anti-FLAG MAb and blotted (IB) with anti-ubiquitin-MAb. There was an increase in ubiquitination of β-arrestin 2 with increasing exposure to 1 mM carbachol. Each blot is representative of two independent experiments. Bottom panel: lysates blotted with anti-FLAG MAb to demonstrate β-arrestin 2 expression levels.