Figure 3

Incomplete glycosylation of mutant PDGFRα. (A) 293FR-PDGFRα stable cell lines were either treated with doxycycline (5 ng/ml) for 10 h or were left untreated (uninduced). Collected total cell lysates were either left untreated or were treated with Endo H or PNGase F in order to remove mannose or complex glycans from glycoproteins, respectively. The different glycosylated forms of PDGFRα were investigated by Western blot analysis using a PDGFRα antibody (B) 293FR-PDGFRα cell lines were treated with doxycycline for 14 h. In addition, each cell line was incubated with three different PDGFR-α inhibitors (G: Gleevec (1 μM), D: Dasatinib (1 μM), S: Sunitinib (1 μM)). The activation and presence of the different PDGFRα proteins were assessed by Western blot analysis. An ERK2 staining is represented as loading control. (C) 293FR-PDGFRα-V561D cells were treated with doxycycline for 14 h. The cells were subsequently incubated with Gleevec (1 μM) for the times indicated and the distribution of the glycosylated bands was analysed by Western blot using a PDGFRα antibody. As a loading control, a tubulin staining was additionally performed.