Nrp1 antibody

Primary antibodies were a rabbitmonoclonal NRP-1 antibody (1:200, Abcam, Cambridge, MA), a mouse monoclonal VEGFR-2 (1:50, Abcam, Cambridge, MA). Primary antibodies were detected by using secondary antibodies of anti-rabbit IgG-TR(Texas Red) (Abcam, Cambridge, MA), anti-mouseIgG-FITC (Abcam, Cambridge, MA), respectively. L02 were cultured on attachment factor-coated slide wells (Sonic Seal Slide Well, Nalge Nunc International). Cells were incubated with primary antibodies overnight at 4°C. Sections were washed three times in PBS, followed by secondary antibody for 1h at room temperature. Samples were analyzed with an inverted fluorescence microscope (Olympus IX51) equipped with an Olympus Qcolor 3 digital camera (Olympus).

Cells were lysed in RIPA containing phosphatase inhibitors and protease inhibitors. Lysates were incubated with the indicated primary antibody or IgG (as a negative control) at 4 °C overnight. The antibody-protein complexes were pulled down with protein A/G agarose beads (Santa Cruz Biotechnology. Santa Cruz, USA) and applied for Western blot analysis with NRP-1 antibody (1:30, Abcam, Cambridge, United Kingdom) and GAPVD1 antibody (2-5 μg/mg lysate, Bethyl Laboratories).
Afterward, the bound proteins were extracted from IP beads and digested into peptides for further LC‒MS analysis. LC‒MS/MS experiments were performed on a Q Exactive Plus mass spectrometer that was coupled to Easy nLC (Thermo Scientific). Then the MS data were analyzed using MaxQuant software version 1.6.0.16. MS data were searched against the UniProtKB database (36080 total entries, downloaded 08/14/2018).

Immunohistochemistry (IHC) staining was performed. All specimens were fixed in 4% formaldehyde solution, embedded into paraffin, and then cut into 4μm sheets. In the next step, the tissue slides were dewaxed with xylene, dehydrated by using gradient alcohol and retrieved antigen by microwave. NRP1 antibody (Abcam, Cambridge, USA) diluted to 1:200. The secondary antibody was labeled with peroxidase and developed with diaminobenzidine. The staining procedure was carried out according to the manufacturer's instructions. The result of immunohistochemistry (IHC) was judged by two experienced pathologists and quantified by using a semi-quantitative comprehensive scoring method based on the intensity of staining and the ratio of stained cells 18 : the immunoreactive score (IRS) depended on the intensity of staining (SI) and the percentage of the positive cell (PP), IRS=SI+PP. SI: 0 points for the colorless, 1 point for the light yellow, 2 points for the brownish yellow, and 3 points for the tan. PP: 0 points for negative, 1 point for positive cells ≤10%, 2 points for 11% to 50%, 3 points for 51% to 100%. IRS: 0 negatives, ≤3 low expressions, > 3 high expressions.