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Figure 3: Immunofluorescence labeling revealing the localization of the PDGF β-receptor upon exposure to PDGF-BB. Serum-starved mouse fibroblasts were stimulated with PDGF-BB and chemically fixed after 15 minutes. Samples represented in (d), (e), and (f) were subjected to a triton treatment after chemical fixation. Subsequently cells were stained with two different antibodies raised against the PDGF β-receptor (green) and stained for F-actin with phalloidin-TRITC (red), similar to Figure 2. Upon PDGF-BB stimulation circular ruffles were induced as detected by staining for F-actin ((b), (e), and (h)). The antibody that recognizes the intracellular domain of the PDGF β-receptor co-localized with F-actin in the dorsal ruffles ((a), arrows). The number of patches with receptors decreased as compared with nonstimulated cells (Figure 3(a) versus Figure 2(a)). In samples that were subjected to a triton treatment after chemical fixation ((d), (e), and (f)), the patches were no longer visible and the intracellular cytoplasmic labeling was more intense (d). Also in these samples the PDGF β-receptor co-localized with F-actin in dorsal circular ruffles ((d), arrow). Immunofluorescence studies with the antibody that recognizes the extracellular domain of the PDGF β-receptor revealed that upon stimulation with PDGF-BB the patches of PDGF β-receptors often disappear (g). In cells that form circular ruffles, the PDGF β-receptor partly localizes in the newly formed circular ruffles ((g), arrow) that were revealed by labeling for F-actin (h). Bar represents 10 μm.