Measurement of ERK1/2 activation.

HEK293 cells inducibly expressing α_2B-AR were cultured in 6-well dishes and transfected with GGA3 siRNA or shRNA for 36 h. The cells were then incubated with tetracycline at 40 ng/ml for 24 h. After starvation for at least 3 h, the cells were stimulated with UK14304 for 5 min. In some experiments, the cells stimulated with phorbol-12-myristate-13-acetate (PMA) at a concentration of 1 μM for 5 min were used as a positive control. To measure extracellular signal-regulated kinase 1/2 (ERK1/2) activation by endogenous α₂-ARs, cells were transfected with GGA3 siRNA for 36 h and then stimulated with UK14304 as above. Stimulation was terminated by addition of 1× SDS-loading buffer. ERK1/2 activation was determined by immunoblotting using phospho-specific ERK1/2 antibodies (Santa Cruz) as described previously (28).