PDGF induced phosphorylation events in cells of different <i>Lrig2</i> genotypes.

<p>Wild-type, heterozygous, or <i>Lrig2</i>-deficient MEFs were serum starved for 24 hours followed by stimulation with 50 ng/ml PDGF-BB for different times. (<b>A</b>–<b>N</b>) Cells were untreated or stimulated with 50 ng/ml PDGF-BB for 10 minutes followed by cell fixation and analysis of the phosphorylation status of respective Pdgfr by <i>in situ</i> proximity ligation assay (PLA). Phosphorylated Pdgfr was visualized using fluorescence (red spots). Cell nuclei were counter-stained with DAPI (blue). (<b>A</b>–<b>F</b>) Representative PLA images of phosphorylated Pdgfrα (red spots) in un-stimulated (<b>A</b>–<b>C</b>) or PDGF-BB stimulated (<b>D</b>–<b>F</b>) cells of the indicated <i>Lrig2</i> genotypes. (<b>G</b>–<b>L</b>) Representative PLA images of phosphorylated Pdgfrβ (red spots) in non-stimulated (<b>G</b>–<b>I</b>) or PDGF-BB-stimulated (<b>J</b>–<b>L</b>) cells of the indicated <i>Lrig2</i> genotypes. (<b>M</b>) Quantification of PLA spots for phosphorylated Pdgfrα. Shown are the means from three independent experiments, including wild-type (<i>Lrig2E12+/+</i>, n=8), heterozygous (<i>Lrig2E12+/-</i>, n=9), and <i>Lrig2</i>-deficient (<i>Lrig2E12-/</i>-, n=6) cell lines from three different litters, with standard deviations indicated by error bars. (<b>N</b>) Quantification of PLA spots for phosphorylated Pdgfrβ. Shown are the means from three independent experiments, including wild-type (<i>Lrig2E12+/+</i>, n=8), heterozygous (<i>Lrig2E12+/-</i>, n=9), and <i>Lrig2</i>-deficient (<i>Lrig2E12-/</i>-, n=5) cell lines from three different litters, with standard deviations indicated by error bars. There were no differences observed in the levels of activated Pdgfrα or Pdgfrβ between cells of different genotypes. (<b>O</b>–<b>Q</b>) Cell lysates from cells that had been untreated or treated with 50 ng/ml PDGF-BB for 15 minutes were analyzed through Western blotting with antibodies against the indicated proteins. (<b>O</b>) A representative Western blot is shown of Akt, Erk1/2, phosphorylated Akt (pAkt), and phosphorylated Erk1/2 (pErk1/2) using cell lysates from cells of the indicated <i>Lrig2</i> genotypes that had been untreated (-) or treated (+) with PDGF-BB. (<b>P</b>) Quantification of pAkt/Akt-ratios for non-stimulated and stimulated cells, respectively. Shown are the means from three independent experiments, including wild-type (n=8), heterozygous (n=9), and <i>Lrig2</i>-deficient (n=5) cell lines from three different litters, with standard deviations indicated by error bars. (<b>Q</b>) Quantification of pErk1/2/Erk1/2-ratios for non-stimulated and stimulated cells, respectively. Shown are the means and corresponding standard deviations as for <b>P</b>.