Expression and autophosphorylation of PDGF-Rβ mutants in PAE cells.

<p>PAE cells were stably transfected with vectors expressing the different PFBC mutations found in <i>PDGFRB</i>. (<u>A</u>) After mRNA extraction, the total expression of human <i>PDGFRB</i> was detected with qPCR. Error bars indicate standard deviation between 3 independent experiments. ND: Not detected. (<u>B</u>) Immunoblot demonstrating the protein expression levels of the different <i>PDGFRB</i> mutants. Stable clones of mutant <i>PDGFRB</i> expressing PAE cells were treated with a proteasomal inhibitor (MG-132), lysosomal inhibitor (chloroquine) or vehicle for 4 hours before lysis. Total levels of PDGF-Rβ were visualized with anti-total PDGF-Rβ (28E1) antibody. The graphs indicate relative expression level of the inhibitor-treated conditions <i>vs</i> control condition for three individual experiments. Error bars indicate standard deviation. *<i>p</i><0.05 compared to basal wild type (WT) expression, ^#<i>p</i><0.05 when comparing inhibitor-treated <i>vs</i> control condition for each mutant. (CD) Autophosphorylation of the PDGF-Rβ mutants. PAE cells stably expressing mutant PDGF-Rβ were exposed to 40 ng/ml of exogenous PDGF-BB for 60 minutes after cooling on ice. A wild-type <i>PDGFRB</i>-expressing construct was used as a positive control, while a kinase dead (KD) variant was used as a negative control. Cell lysates were adjusted to yield a comparable amount of PDGF-Rβ signal. (C) Representative western blot demonstrating autophosphorylation of the different mutants on four residues. (D) Quantification of PDGF-Rβ autophosphorylation signal from tyrosine residues 751, 771, 1009 and 1021 using phospho-specific antibodies. Signals were normalized over total levels of PDGF-Rβ protein, and expressed as a percentage of wild-type PDGF-Rβ autophosphorylation. The graph represents the averaged results from the 4 tyrosine residues that were assessed in three independent experiments. *<i>p</i><0,05 when compared to the positive control (WT PDGF-Rβ).