Fig. 5

Internalization of PDGFRα from the cell surface is not affected by mutation of lysine residue 917. (a) PAE cells were transfected with WT or K917R mutant PDGFRα and serum-starved for 24 h, followed by stimulation with 20 ng/ml PDGF-AA for indicated time periods. Cell surface proteins were then biotinylated. After lysis of cells, biotinylated proteins were collected on streptavidin-agarose beads. Adsorbed proteins and total cell lysates were subjected to SDS-PAGE and immunoblotted with antibodies against PDGFRα, transferrin receptor (TfR) or Alix. Alix was used as a loading control. (b) The expression level of PDGFRα remaining on the cell surface compared with transferrin receptor (TfR) from three independent repeats was quantified. The ratio at 0 min of PDGF-AA stimulation was set at 1. (c) Expression of WT or K917R mutant PDGFRα in a tet-inducible PAE cell line was induced with doxycycline and assessed as described in panel a. (d) The PDGFRα levels on the cell surface relative to TfR on the cell surface from three independent repeats was quantified. The ratio of induced and unstimulated cells was set as 1. The immunoblots were cropped for clarity. Full length blots are presented in Figure S1