Fig. 7

Mutation of lysine 917 affects PDGFRα activation of PLCγ and STAT3 and cell proliferation. (a) PAE cells were transiently transfected with WT or K917R mutant PDGFRα, serum-starved and stimulated with 20 ng/ml PDGF-AA for indicated time periods. Expression levels of phosphorylated PLCγ (b), STAT3 (c), Akt (d) and ERK1/2 (e) and total proteins were determined using antibodies against phosphorylated PDGFRα (pY849), PLCγ (pY783), STAT3 (pY705), Akt1/2/3 (pS473), ERK1/2 (pThr202/pThr204), and their non-phosphorylated counterparts, as well as α-tubulin as a loading control. The immunoblots were cropped for clarity. Full length blots are presented in Figure S1. (b-e) Quantification of phosphorylated proteins relative to total proteins. Phosphorylation at 30 min of stimulation with PDGF-AA was set as 1. The experiment is a representative one out of three experiments performed with similar results. (f, g) Tet-inducible PAE cells were seeded into 96 well plates, incubated in Ham’s F-12 media supplemented with 1% FBS, induced or not with doxycycline, and then stimulated with 0, 1, 5, 10 or 20 ng/ml of PDGF-AA (f) or PDGF-BB (g) for 72 h. Cells incubated in media with 10% FBS were used as a positive control. WST-1 was added into the wells for 4 h and the absorbance of cells incubated in 1% FBS without doxycycline and PDGF ligands was set as 1. The results from four independent repeats are plotted. The statistically significant difference between WT and K917R mutant PDGFRα tet-inducible PAE cell lines was determined by unpaired, two-tail student t-test. *p < 0.05