NRP1 promotes FN-induced PXN Y118 phosphorylation. (A and B) HDMECs transiently transfected with control, NRP1 or VEGFR2 siRNA were plated on FN for the indicated times and immunofluorescently labeled (A) for pPXN Y118 (green) together with phalloidin (red) and DAPI (blue; bar, 20 µm) or immunoblotted for the indicated proteins (B). The single channel for pPXN staining is shown on the right side of A. (C) pPXN Y118 levels in immunoblots from 4 independent experiments were quantified as pixel intensity relative to GAPDH and values expressed as mean fold change ± SD in si-VEGFR2 or si-NRP1 relative to control siRNA transfected cells. *, P < 0.05, Student’s t test. (D) To examine if NRP1 forms a complex with pPXN in FN-stimulated ECs, HDMECs were detached in serum-free medium (nonadherent, NA) or plated on FN for the indicated times and then immunoprecipitated with control IgG or NRP1 antibodies, followed by immunoblotting for NRP1 and pPXN Y118. The immunoblot is representative of 4 independent experiments. (E) Single confocal scans of HDMECs plated on FN for 45 min and then immunofluorescently labeled with antibodies specific for human NRP1 or pPXN Y118 and counterstained with DAPI. Bar, 10 µm. A higher magnification of the area indicated with a dotted square is shown in the bottom row, and single channels for NRP1 and pPXN are shown adjacent to the triple stains at low and high magnification. Arrowheads indicate examples of partial colocalization. (F) HDMECs were transfected with pCDNA3.1 encoding wild-type mouse NRP1 or mutant mouse NRP1D320A, plated on FN for 60 min, and then labeled with antibodies specific for mouse NRP1 and pPXN and counterstained with DAPI. The single channel for pPXN staining is shown on the right. Cells expressing murine NRP1 (white arrowheads) up-regulate pPXN. Note that the antibody does not detect endogenous human NRP1 in untransfected cells (clear arrowheads). Bar, 20 µm.
