Figure 8. K5 mediates rapid internalization of RTKs from the surface leading to increased signaling.

(A) Equal numbers of the indicated THP-1 lines were mock or Dynasore (80 µM) treated for an hour at 37°C. Cells were then released at 37°C in serum free media for 0, 5, 10 or 30 minutes. At the indicated time points, cell aliquots were fixed and stained for surface levels of Flt-3, Flt-4 and PDGFR-ß followed by flow cytometric analysis. Representative experiments for each of the three receptors demonstrating the relative change in the expression of each RTK normalized to the fluorescence (geometric means) of mock treated samples is shown as line graphs, while the overall endocytosis rate of the receptor tyrosine kinases over the 30 minutes following dynasore release normalized to the time 0 point is shown as a bar graph with standard deviations. Line graph results are representative of at least three experiments (B) Equal numbers of vector- and K5 wt-expressing THP-1 were seeded in media containing 2% FBS. Following treatment with DMSO (mock) or Dynasore (80 µM) for 60 min and release in Dynasore-free medium at 37°C for 0, 30 or 60 minutes normalized whole cell lysates were prepared and subjected to western blot (WB) with an anti-phospho-tyrosine (4G10) (pTyr) antibody.