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. 2010 Feb 1;30(7):1593–1606. doi: 10.1128/MCB.00038-09

FIG. 10.

FIG. 10.

The C terminus of LPP3 is required for p120ctn binding and formation of branching point structures. (A) Schematic of LPP3 retroviral constructs pLNCX2-HA-WT-hLPP3 (wild type), pLNCX2-HA-N-Δ-hLPP3 (lacking N-terminal cytoplasmic segment), and pLNCX2-HA-C-Δ-hLPP3 (lacking C-terminal cytoplasmic segment). The second extracellular loop (II nd extracellular loop) and transmembrane segments (segments 1 to 6) are shown. (B and C) ECs expressing pLNCX2-HA-WT-hLPP3(1), pLNCX2-HA-N-Δ-hLPP3(2), and pLNCX2-HA-C-Δ-hLPP3(3) were detached with 3 mM EDTA, washed, and subjected to cell surface biotinylation as described in Materials and Methods. Clarified cell extracts were immunoprecipitated with anti-HA antibody and analyzed by immunoblotting with anti-p120ctn MAb (B) and streptavidin-HRP (C). (D to J) To deplete endogenous LPP3, ECs at 50% density were infected with LPP3sh(c) (L) for 6 h and allowed to recover. After 12 h, ECs were infected with the indicated constructs (constructs 1 to 3) for 6 h, allowed to recover for 4 h, and then subjected to branching point, cell proliferation, and cell migration assays. (D and G to J) Branching point assay in three-dimensional type I collagen matrix. (D) Quantification of branching points. After 24 h, the number of branching points were scored. Data are expressed as percentages of branching points (n = 8). *, P < 0.05 versus L. (G to J) Representative images of branching point formation in response to reexpression of indicated constructs (constructs 1 to 3). Experiments were repeated three times using triplicate wells each time. The black arrows point to branching points. Magnification, ×200. Bar, 200 μm. (E) Percent bromodeoxyuridine (BrdU) incorporation of ECs in response to reexpression of indicated constructs (constructs 1 to 3). The cells were cultured on coverslips coated with 0.2% gelatin for 16 h, fixed, and stained. BrdU-positive cells were enumerated and expressed as percent cell proliferation (n = 8). *, P < 0.05 versus L. (F) Percent cell migration in response to reexpression of the indicated constructs (constructs 1 to 3). P < 0.05 versus L. (K to M) Whole-cell lysates from cells expressing constructs 1 to 3 were subjected to WB analyses with the antibodies indicated. All blots shown are representative of at least three separate experiments.