Figure 5

Blocking PDGFRβ dimerization inhibits ERK1/2 activation in PDGF-BB-treated, but not in dopamine-stimulated CHO-K1 cells. (A) CHO/DRD4-PR cells were pretreated with 4 μg/mL of either GST or GST-PDGFRβ immunoglobulin domain 4 (GST-Ig4β) fusion proteins for 20 min at 37°C to prevent PDGFRβ dimerization and were then stimulated with 1 μM dopamine or 10 ng/mL PDGF-BB for 5 min. Lysates were taken for western blotting with phospho-ERK1/2 antibody (upper blot). The same blot was stripped and reprobed for total PDGFRβ to demonstrate equal loading of all lanes (lower blot). (B) Pre-treatment with 100 nM wortmannin for one hour abolished the dopamine-mediated ERK1/2 signal following PDGFRβ dimerization block with GST-Ig4β. The same blot was stripped and reprobed for β-tubulin to demonstrate equal loading of all lanes (lower blot). The bar graphs show the densitometric measurement of the relative signals from phospho-ERK1/2 over two to four experiments, and the quantities were given as mean ± SEM.