Fifty cells from three impartial experiments were analyzed. for the sorting nexin family that regulates intracellular trafficking and recognized sorting nexin 9 (SNX9) as a novel angiogenic factor in human umbilical vein endothelial cells (HUVECs). SNX9 was essential for cell distributing around the Matrigel, and tube formation that mimics in vivo angiogenesis in HUVECs. SNX9 depletion significantly delayed the recycling of integrin 1, an essential adhesion molecule for angiogenesis, and reduced the surface levels of integrin 1 in HUVECs. Clinically, we showed that SNX9 protein was highly expressed in tumor endothelial cells of human colorectal malignancy tissues. High\level expression of SNX9 messenger RNA significantly correlated with poor prognosis of the patients with colorectal malignancy. These results suggest that SNX9 is an angiogenic factor and provide a novel target for the development of new antiangiogenic drugs. for 10?min at 4C. The resultant supernatants were incubated with streptavidin magnetic beads (Dynabeads M\280; Invitrogen) for 1?hr at 4C. The beads were washed with IP buffer three times followed by the collection of proteins with SDS buffer without 2\mercaptoethanol. The total and biotinylated integrin 1 were detected by western blot analysis using the TS2/16 antibody. 2.11. Integrin 1 uptake and recycling assays The internalization and recycling assays of integrin 1 were performed as explained previously (Arjonen, Alanko, Veltel, & Ivaska, 2012). Briefly, integrin 1 around the cell surface of HUVECs was labeled with Alexa488\conjugated TS2/16 antibody in the growth\EBM\2 medium made up of 30?mM Hepes (pH 7.6) on ice for 1?hr. Cells were then washed with ice\chilly PBS and the medium was replaced with fresh growth medium made up of 30?mM Hepes (pH 7.6). For the internalization assay, the cells were incubated at 37C with 5% CO2 for the indicated time\point. After the internalization, the cells were put on the ice and the fluorescence around the cell surface was quenched by adding anti\Alexa488 antibody and incubating on ice for 1?hr. To monitor the recycling of integrin 1, labeled integrin 1 around the cell surface was allowed to internalize for 1?hr at 37C with 5% CO2 followed by quenching of the surface integrin 1. Cells were incubated again at 37C with 5% CO2 for the indicated time\point. After incubation, the surface fluorescence transmission of integrin 1 was quenched again. For imaging, the cells TD-106 FLJ42958 were fixed with 4% PFA in PBS for 30?min at room heat. The fluorescence intensity of Alexa488 excluding the background fluorescence intensity was quantified with ImageJ (NIH). The fluorescence intensities were normalized against the total surface staining (at 0?min before quenching, for the uptake assay) or total internalized staining (for the recycling assay). 2.12. Transferrin uptake and recycling assays The internalization and recycling assays of transferrin were performed as explained previously (Lee et al., 2015). For the uptake assay, HUVECs were serum\starved in EBM\2 for 30?min at 37C. The cells were then incubated with 50?g/ml of Alexa488\transferrin (Molecular Probes) in 0.15% serum\containing EBM\2 for 5 or 10?min at 37C. The cells were then chilled on ice and incubated in acid\wash buffer (20?mM sodium\acetate TD-106 buffer; 1?mM CaCl2; 150?mM NaCl; pH 4.8) on ice for 5?min to remove Alexa488\transferrin in the PM. For the recycling assay, HUVECs were incubated in 0.15% serum\containing EBM\2 for 30?min at 37C followed by incubation in 0.15% serum\containing EBM\2 containing 50?g/ml Alexa488\transferrin for 1?hr at 37C. After washing with ice\chilly PBS, the cells were incubated in the acid\wash buffer on ice for 5?min to remove the surface\bound Alexa488\transferrin. Cells were washed with ice\chilly PBS and chased in growth\EBM\2 medium made up of 400?g/ml unlabeled human holo\transferrin (Thermo Fisher TD-106 Scientific) at 37C with 5% CO2. For imaging, the cells were fixed with 4% PFA in PBS at room heat for 30?min. The fluorescence intensity of Alexa488 excluding the background fluorescence intensity was quantified with ImageJ (NIH). 2.13. Distributing and network formation around the Matrigel HUVECs were collected by treatment with trypsin for 1?min followed by seeding around the Matrigel TD-106 basement membrane (BD Matrigel? Basement Membrane Matrix Growth Factor Reduced; BD Biosciences, Tokyo, Japan). The cells were then incubated in the growth\EBM\2 medium for 1?hr or 12?hr at 37C with 5% CO2. The cells were fixed and F\actin was stained with rhodamine\conjugated phalloidin. The cell size and network length were measured with ImageJ (NIH, Bethesda, MD). 2.14. Tube formation assay Collagen I gel was reconstituted by.