Vascular endothelial growth factor receptor 2 (VEGFR-2) is an endothelial cell-specific receptor that has inherent tyrosine kinase activity. The binding of the Vascular endothelial growth factor (VEGF) ligand to its receptor plays an important role in angiogenesis, a process by which new capillaries are formed. Enhanced angiogenesis is associated with a number of diseases including cancer. Your goal is to develop a homogeneous in vitro TR-FRET kinase assay for VEGFR-2 (purified protein) in an attempt to identify small molecules that inhibit kinase activity and in turn modulate angiogenesis.   Based on this background, please answer the following questions  How would you apply a TR-FRET assay to measure the in vitro kinase activity of VEGFR-2? Describe in steps the assay design and potential readout outcome.

You want to understand how the density of P-selectin in blood vessel walls affects the rolling interactions of neutrophils when they are subjected to hydrodynamic drag forces in the blood. You introduce P-selectin into a synthetic lipid bilayer and attach it to a glass slide mounted in a flow chamber. This arrangement allows you to measure neutrophil attachment at different densities of P-selectin and at different flow rates. At high densities, from 30 to 400 P-selectin molecules per µm², the neutrophils attached to the membrane and rolled very slowly and jerkily in the direction of the flow (see cell 1 in the figure). At the same flow rate, but at a density of 15 P-selectins per µm², the neutrophils behaved differently: they either moved at the flow rate of the medium or were transiently tethered-horizontal lines marked by t-before moving again (cell 2 in the figure). Neutrophils showed this same behavior at a density of 1 P-selectins per μm² and half the flow rate: moving at the flow…

Cystic fibrosis (CF) is a genetic disorder caused by mutations in the CFTR (cystic fibrosis transmembrane-conductance regulator) gene, which encodes the CFTR protein. The cells of the lining of the lungs contain high levels of the CFTR protein in their membranes. Normally, CFTR contains a chloride ion channel, which permits chloride ions to pass from inside the cells to the cells’ surfaces. The surface chloride ions are able to attract water in the lungs, allowing for the formation of a water layer on the cells’ surfaces. This thin water layer is necessary to allow the cilia, the tiny hairs on the surface of the lining cells to move back and forth easily. This motion permits the removal of mucus from the lungs. A particular error in the CFTR gene results in the replacement of the amino acid Phe with Ser at residue 508 of the CFTR protein. Individuals afflicted with CF most often experience the presence of thick, dry and sticky mucus in the lungs, leading to chronic infections and…

Rho activation is preceded by association of a Rho regulator to regions of the plasma membrane just “above” where the contractile ring will form. Which regulator (Rho GAP or Rho GEF) would you predict would be localized to this region of the cell? Briefly explain your reasoning.