Fig. 5.
Platelet-derived growth factor (PDGF)-A promotes interactions between PDGF receptor (PDGFR)-α and neuropilin-1 (NRP1). Distal pulmonary mesenchymal cells from wild-type C57BLK/6J mice at postnatal day 12 were isolated by differential adherence to tissue culture plastic (which selects for fibroblasts and pericytes) and cultured. Confluent cells that had been exposed to PDGF-A or remained unexposed were used to isolate membrane lipid rafts (MLRs), and samples were fractionated in a discontinuous gradient of OptiPrep. Gradient fractions are numbered from the least-dense fraction 1 [containing buoyant Triton X-100-resistant membrane (cholesterol-rich fraction)] to the more-dense fraction 10 (cytosolic proteins). A: caveolin is retained in MLRs, whereas exposure to PDGFR-A shifts NRP1 and PDGFRα to more-dense fractions. Immunoblot is representative of cell isolations from 2 additional litters. B: freshly isolated distal pulmonary mesenchymal cells obtained from 4 mice from separate litters were stained for PDGFRα (CD140a), NRP1, CD9 (marker of caveolin-rich MLRs), or CD71 (marker of clathrin-coated vesicles), briefly exposed to Triton X-100 (TX-100) or remained unexposed (Untr), fixed, and then analyzed by fluorescence-activated cell sorting. A majority of CD140a and NRP1 antigens were resistant to Triton X-100, indicating that they reside in MLRs. C and D: pulmonary mesenchymal cells isolated from Pdgfrαtm11(EGFP)Sor/J mice adhered to fibronectin-coated glass. Duolink proximity ligation assay was used to quantify cellular granules in cultures treated with PDGF-A for 0−30 min. Values are means ± SE; n = 4 separate cell isolations of granule counts from 3 separate experiments for comparisons between 2 treatment conditions. *P < 0.05 [by 1-way ANOVA (C) and t-test for paired variables (D)].