Sc 74403

Primary antibodies used were mouse-anti-NDPK-B (MC-412, Kamiya, Seattle, WA, USA, 1:1000), rabbit-anti-NDPK-A (sc-343, Santa Cruz, Heidelberg, Germany, 1:500; detects both NDPK-A and NDPK-B in mouse retinae), mouse-anti-Ang-2 (sc-74403, Santa Cruz, Heidelberg, Germany, 1:500), mouse-anti-O-GlcNAc (ab-2739, Abcam, Cambridge, UK, 1:1000), rabbit-anti-OGA (SAB4200267, Sigma, Munich, Germany, 1:1000), rabbit-anti-OGT (O-6264, Sigma, Munich, Germany, 1:1000), rabbit-anti-N1-phosphohistidine (MABS1330, Millipore, Darmstadt, Germany, 1:1000), mouse-anti-γ-tubulin (T6557, Sigma-Aldrich, Munich, Germany, 1:2000), rabbit-anti-GFAT (obtained in cooperation with Weigert, Tübingen), and sheep-anti-pGFAT (MRC-PPU s343c) for immunoblotting. The secondary antibodies used were rabbit anti-mouse peroxidase (A9044, Sigma-Aldrich, Munich, Germany, 1:20,000), goat-anti-rabbit peroxidase (A9169, Sigma-Aldrich, Munich, Germany, 1:40,000), and donkey-anti-sheep peroxidase (Sigma-Aldrich). qPCR primers were obtained from Applied Biosystems, ThermoFischer: GFAT Hs00899865_m1, OGA Hs00201970_m1, OGT Hs00269228_m1, and 18S Hs03003631_g1. Gelatin from porcine skin (48720, Fluka, Bucharest, Romania) was used as a 1% solution in PBS. Thiamet G (TMG; SML0244, Sigma-Aldrich, Germany) treatment was given at 10 µM for 24 h.

Ninety-six-well plates were coated with 50 μL/well growth factor-reduced Matrigel (Corning Life Sciences, Tewksbury, MA) and incubated for 1 hour at 37 °C for gel solidification. After starvation in endothelial basal medium (CC-3121, Lonza) supplemented with 0.1% (w/v) bovine plasma albumin for 16 hours, HUVECs or HMVECs were trypsinized, resuspended in diluted plasmas, and seeded at 20,000 cells/well. Pooled plasmas, with or without Ang2 removal, from either healthy controls or patients were diluted 1:3 with EGM. To remove Ang2, the plasmas were treated with Ang2 specific antibody (sc-74403, Santa Cruz Biotechnology, Dallas, TX) conjugated M-270 Epoxy beads prepared using Dyna beads Antibody Coupling Kit (14311D, Life Technologies, Carlsbad, CA). Concentrations of the pooled samples, with or without Ang2 removal, were measured with enzyme-linked immunosorbent assay. Images (50× magnification) of tubular network were taken with a Leica Microsystems 4000B microscope equipped with a RTKE Spot camera (Diagnostic Instruments, Sterling Heights, MI) 12 hours after cell plating. Tube branching points presented by the image were counted with ImageJ (National Institutes of Health, Bethesda, MD). Experiments were performed in quadruplicate and repeated twice.

Proteins from HUVECs and rMCs were extracted in RIPA buffer using a previously described method [25 (link)]. The primary antibodies used were: VEGFR2 (55B11 Cell Signaling, 1:1000); Ang2 (SC74403 Santa Cruz, 1:500); Tubulin (T6557 Sigma–Aldrich, 1:2000); GlcNAc (ab2739 Abcam, 1:1000); GFAP (Z0334 Dako, 1:5000); VEGF (ab16154 Abcam, 1:2000), diluted in Tris-buffered saline containing 0.1% Tween20 (TBST). Secondary antibodies against mouse (Rabbit anti-mouse peroxidase, A-9044 Sigma–Aldrich) and rabbit (Goat anti-rabbit peroxidase, A-9169 Sigma–Aldrich) were diluted 1:20,000 in TBST. The obtained images were quantified using ImageJ.