Fig. 3

Mutation of lysine residue 917 does not affect the steady state level or ligand-induced degradation of PDGFRα. (a) WT or K917R mutant PDGFRα were transfected in COS-7 cells, followed by treatment with 50 µg/ml cycloheximide for 0, 1, 2, 4 and 6 h. The cells were then lysed and immunoblotted with antibodies against PDGFRα and Alix as a loading control. (b) The expression of PDGFRα was quantified. WT or K917R mutant PDGFRα levels at 0 h treatment of cycloheximide was set at 1. (c-f) WT or K917R mutant PDGFRα were transiently transfected into PAE cells (c) or induced with doxycycline in PAE-PDGFRα tet-inducible cell line (e), followed by serum-starvation for 24 h and treatment with 50 µg/ml cycloheximide for 1 h. Cells were then stimulated with PDGF-BB for 0, 0.5, 1 or 2 h, after which the amount of PDGFRα was examined by immunoblotting, and quantified relative to the levels of Alix or α-tubulin which served as loading controls (d, f). The results of three independent experiments were quantified. Expression levels of WT or K917R mutant PDGFRα without PDGF-AA stimulation were set at 1. (g) Tet-inducible PAE cells were treated with doxycycline for 48 h to induce the expression of WT or K917R mutant PDGFRα and starved, followed by incubation with 25 µM chloroquine (CQ) or 1 µM bortezomib (BTZ) for 4 h; DMSO served as a control. Cells were then stimulated with 20 ng/ml PDGF-AA for 45 min and lysed for immunoblotting. (h) Quantification of the results of panel g. The level of WT PDGFRα without inhibitor treatment and PDGF-AA stimulation was set as 1. All experiments were performed three times or more except for panels a and g which were performed two times. The immunoblots were cropped for clarity. Full length blots are presented in Figure S1