Figure 4.

The proteosomal inhibitor lactacystin inhibits the β-arrestin 2 mediated agonist-promoted down-regulation of the M₂ mAChR in MEFwt. MEFwt cells were transfected with HA-M₂ mAChR and, after 24 hr cells were incubated for 20 min with or without 10 μM lactacystin then treated with 1 mM carbachol for 4 hr. Lactacystin inhibits the β-arrestin 2 mediated down-regulation of the M₂ mAChR receptor in response to agonist. Down-regulation was determined using [³H]-QNB binding in crude membranes as described in methods. Data are expressed as [³H]-QNB bound (fmol/mg total protein) and are presented as mean ± standard deviation from three independent experiments with duplicate data points. Statistical analysis was performed using a paired t-test, ** indicates p ≤ 0.001 (compared to paired untreated control), ns indicates not significant.