Fig. 1.

High glucose increases the association of PI 3 kinase with the PDGFRβ leading to its phosphorylation. (A, B and E) Mesangial cells were incubated with high glucose (HG, 25 mM glucose) for the indicated time periods. As control (0 h), 20 mM mannitol plus 5 mM glucose was used as described in the Materials and methods section. Equal amounts of cell lysates were immunoblotted with phospho-p85 (Tyr-458), p85, phospho-PDGFRβ (Tyr-857), phospho-PDGFRβ (Tyr-740), phospho-PDGFRβ (Tyr-751) and PDGFRβ antibodies as indicated. (C, F and G) Mesangial cells were treated with 0.1 μM JNJ prior to incubation with high glucose (HG) and normal glucose (NG) for 24 h. In panel C, the cell lysates were immunoblotted with the indicated antibodies. In panels F and G, the cell lysates were immunoprecipitated with PDGFRβ (panel F) or with p85 PI 3 kinase subunit (panel G) antibodies. The immunoprecipitates were immunoblotted with p85 (panel F) or PDGFRβ (panel G) antibodies, respectively. (D and H) Mesangial cells were transfected with scramble siRNA or siRNAs against PDGFRβ (panel D) or with vector or PDGFRβ (Y740/Y751F) mutant as described in the Materials and methods section. The transfected cells were incubated with high glucose (HG) or normal glucose (NG) as described above. The cell lysates were immunoblotted with the indicated antibodies.