Figure 1.

Expression of LPP3 protein in monolayer ECs. Confluent ECs (passage 4) were starved for 6 h in conditioned medium (M199 supplemented with 0.2% BSA + 1× ITS), and then stimulated with VEGF¹⁶⁵ (100 ng/ml) or bFGF (20 ng/ml) for various durations, as indicated. Cells were solubilized, clarified by centrifugation, and the protein concentrations were determined. Samples were subjected to SDS-PAGE and analyzed by immunoblotting with: (A) Rabbit anti-LPP3-c-cyto polyclonal antibody (2.0 μg/ml). Anti-LPP3-c-cyto antibody detects unprocessed LPP3 protein that appears as ~36 kDa, and two major polypeptides migrate below the ~52 kDa molecular weight marker, LPP3 polypeptides are indicated by arrowheads; (B) Anti-PCNA monoclonal antibody (0.5 μg/ml). (C) Anti-Fak monoclonal antibody (1.0 μg/ml). (D) Cell surface biotinylation of intact cells and immunoprecipitation analysis. K562 (lane 1, unstimulated), HUVECs (lane 2, stimulated with VEGF, 100 ng/ml 6 hours), and HUVECs (lane 3, stimulated with bFGF, 20 ng/ml for 6 h) subjected to cell surface biotinylation, lysed in RIPA buffer, clarified by centrifugation and immunoprecipitated using anti-LPP3-c-cyto (5 μg) antibody. Immunocomplexes were resolved by 10% SDS-PAGE under reducing condition and analyzed by ligand blotting with streptavidin-HRP (1:10000). Data shown are representative of those obtained in at least three separate experiments, with similar results. (E) De-N-glycosylation of LPP3 protein. Monolayer HUVECs (passage 4) were stimulated with VEGF¹⁶⁵ for 6 h and subjected to cell surface biotinylation, lysed in RIPA buffer, clarified by centrifugation, and immunoprecipitated by either rabbit IgG (control) or anti-LPP3-c-cyto antibodies, as indicated. Following immunoprecipitation with anti-LPP3-c-cyto antibodies, the contents were equally divided into two tubes. One tube was left untreated, and the second tube treated with N-Glyganase (50 units of PNGaseF) enzyme at 37°C for 3 h. Immunocomplexes were analyzed by ligand blotting with streptavidin-HRP (1:10000). Arrowheads indicate LPP3 polypeptides. Arrows indicate unknown polypeptides. Molecular weights are given in kiloDaltons (kDa). Data shown are representative of those obtained in at least three separate experiments, with similar results.