Figure 6.

Regulation of PDGFRA expression by ErbB4 isoforms. (A) NR6 transfectants were cultured for 8 h in the absence of serum, stimulated for 2 h with or without 50 ng/ml NRG-1, and analyzed for PDGFRA mRNA expression by RT-PCR. (B) NR6 transfectants were treated with or without 10 μM AG 1478 for 8 h in the absence of serum. PDGFRA mRNA expression was analyzed by RT-PCR. (C) Cells were cultured overnight in the presence or absence of 10% FCS. PDGFR-α protein expression was analyzed by Western blotting with anti-PDGFR-α antibody. n.s., nonspecific band also present in the vector control lanes. (D) Cells were starved overnight without serum and stimulated for 10 min with 0 or 50 ng/ml PDGF-BB. PDGFR-α tyrosine phosphorylation was analyzed by immunoprecipitation with an anti-PDGFR-α antibody followed by Western blotting with an anti-phosphotyrosine antibody. Membrane was reblotted with an anti-PDGFR-α antibody. (E) NR6 transfectants were plated onto 24-well plates. The next day the medium was replaced with serum-free medium containing or not 20 μM of PDGFR inhibitor AG 1296 or 50 ng/ml PDGF-BB. Adherent cells were counted at indicated time points with hemocytometer. *p < 0.05 for a difference between AG 1296– or PDGF-BB–treated and control cells. (F) NR6 cells expressing JM-a CYT-2 were cultured for 16 h in the presence or absence of 20 μM AG 1296 in serum-free medium. PDGFR-α tyrosine phosphorylation was analyzed by immunoprecipitation with an anti-PDGFR-α antibody followed by Western blotting with an anti-phosphotyrosine antibody. Membrane was reblotted with an anti-PDGFR-α antibody. ErbB4 tyrosine phosphorylation was analyzed by Western blotting with a phospho-specific anti-ErbB4 antibody followed by reblotting with an anti-ErbB4 antibody.