Fig. 3.

Y288C protein is constitutively active at ER and Golgi. a MCF10A cells stably expressing PDGFRA WT (right) or Y288C (left) were serum-starved for 16 h and stimulated with PDGF-BB (50 ng ml^-1) for 15–60 min. Whole cell lysates were resolved by SDS-PAGE and blotted with indicated antibodies to analyze for the activation of PDGFRA and different downstream signaling pathways. Corresponding total proteins were used as loading controls. b Relative cell proliferation of MCF10A parental, PDGFRA WT or Y288C expressing cells in the absence of serum of three independent replicates was assessed for 4 days (exponential growth equation). c Protein lysate of MCF10A expressing PDGFRA WT or Y288C were resolved by SDS-PAGE in the presence or absence of β-mercaptoethanol. Monomeric and dimerized PDGFRA were detected by anti-PDGFRA antibody, and ERK2 was used as loading control. The band intensities of monomers and dimers from three independent experiments were measured by densitometry and normalized to ERK2. The levels of PDGFRA monomers and dimers were presented as mean ± SD relative to WT. The statistical significance of dimer vs the corresponding monomer was assessed by Student’s t-test. d Y288C receptor dimerization in ER and Golgi was analyzed by western blot of the OptiPrep gradient ultracentrifugation resolved membrane fractions in the absence of reducing agent. e Homology model of the Ig-like domain 3 of PDGFRA predicted a hydrogen bond formed between the carboxylate group of D284 and the hydroxyl group of Y288. **p < 0.01; ***p < 0.001