… 37C with occasional shaking followed by filtering through a 40-μm nylon mesh. The cells were
then centrifuged at 94g for 5 minutes at 4C, … ) for 30 minutes at 4C in an overhead shaker. …

… ml fibrinogen solution clotted with 1 U thrombin [Sigma-Aldrich] for 20 min at 37 C). The
second layer of fibrin was clotted for 1 hr at 37 C. C3H10T1/2 cells (250,000 cells/well), in EBM-2 …

Neuropilin 1 (NRP1) plays an important but ill-defined role in VEGF-A signaling and
vascular morphogenesis. We show that mice with a knockin mutation that ablates the NRP1 …

… Lymphatic sprouts strongly express the Nrp2 receptor, and we have previously shown that
LEC sprouting requires VEGF-C binding to a VEGFR3-Nrp2 complex. Site-specific expression …

… For immunoprecipitation, extracts were incubated with an antibody against VEGFR-2 and
protein-G-Sepharose overnight at 4 C. The Sepharose beads were spun down and washed …

… Cells were left at 4C overnight or at −20C for 2 hours. Cells were then collected by centrifugation
and resuspended in 500-μL PBS for 30 minutes. The cells were centrifuged again and …

… 1 A) and dried overnight at 55C. Evans Blue was extracted by incubation in formamide at
55C overnight and quantified by spectrophotometry at 620 nm after subtraction of background …

… C, Western blot analysis of baculovirus recombinant Sema3G using the S1 antiserum and
the myc-antibody identified different forms of the protein after furin proprotein convertase …

… 4C then washed 3× with phosphate-buffered saline (PBS). Tissues were permeabilized and
blocked overnight at 4C … Tissues were then stained with primary antibodies overnight at 4C …

… Another class of permeability-inducing NRP1 ligands is the C-end rule (CendR) peptide
family (29). The presence of a specific RxxR motif at the C-terminal end of these peptides …