Fig. 3
Gi protein is involved in ERK1/2 activation, but Gβγ and arrestins are not. a – FFA1-HEK293 cells were transiently transfected with pCDNA3.1 vector or β-adrenergic receptor kinase COOH domain (495-689aa; βARK-CT) or the Gα subunit of transducin. Both βARK-CT and Gα transducin are scavengers of Gβγ-subunits. The cells were then serum-starved for 24 h and stimulated with 10 μM LA for the indicated periods. b – FFA1-HEK293 cells were transfected with specific arrestin siRNAs or a nonspecific control siRNA. 72 h after transfection, the cells were harvested and equal amounts of total cellular lysate were separated by 10% SDS-PAGE, transferred to a PDVF membrane, and incubated with anti-arrestin antibody. Blots were stripped and reprobed for α-tubulin to control for loading. c – After transfection with specific arrestin siRNAs or nonspecific control siRNA, cells were stimulated with 10 μM LA for the indicated times and immunoblotted using monoclonal anti-phospho-MAPK E10 (Thr-202/Tyr-204). The bolts were stripped and reprobed for total ERK to control for loading. d – FFA1-HEK293 cells were cultured in serum-free DMEM with or without 100 ng/ml PTX for 12 h. The next day, the medium was removed and fresh serum-free DMEM with or without PTX was added for 1 h, and the cells were then stimulated with LA for the indicated times. The data shown are representative of at least three replicate independent experiments. Error bars represent the SEM for three replicates. Data were analyzed using Student’s t-test. ***p < 0.001