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. 2016 Nov 24;6:37708. doi: 10.1038/srep37708

Figure 3. PorZ is located on the cell surface of P. gingivalis.

Figure 3

(a) Wild-type P. gingivalis cells (W83) were proportionately fractionated into whole cell extract (WC), periplasm (PP), cytoplasm (CP), cell envelope (CE), outer membrane (OM), inner membrane (IM) and growth medium (Med), and subsequently analyzed by Western blotting using mouse polyclonal anti-PorZ and anti-Rgp antibodies and mouse monoclonal anti-Kgp antibodies. Streptavidin conjugated to horseradish peroxidase was used to detect MmdC, a biotinylated IM-associated protein. Presence or absence of full-length PorZ (81-kDa band) and other proteins is indicated. (b) Wild-type cells were probed with monoclonal anti-PorZ antibodies and labeled with immunogold to visualize the cellular location of PorZ (black arrowhead) in electron microscopy (bar = 100 nm). (c) Dot blot analysis of intact and lyzed wild-type (W83), ΔPorZ, ΔPorU and ΔPorN cells using mouse monoclonal anti-PorZ antibodies (mAb), mouse polyclonal anti-PorZ antibodies (pAb) or streptavidin conjugated to horseradish peroxidase. Flow cytometry analysis showing the surface exposure of (d) PorZ in wild-type cells (W83) with anti-PorZ pAb; (e) RgpB in wild-type cells (W83) with monoclonal anti-RgpB antibodies (positive control); (f) MmdC in wild-type cells (W83) and (g) in ΔPorZ cells with streptavidin-Alexa Fluor 488 conjugate; (h) PorZ in in trans porZ-complemented ΔPorZ (PorZ+) cells with pAb and (i) PorZ in ΔPorZ cells with anti-PorZ pAb. Isotype negative controls are in blue and immunoprobed cells in red; the histograms shown are representative of three independent experiments. (j) Presence of full-length PorZ (lane 1) detected by Western blot using anti-PorZ pAb on wild-type cells washed with PBS and suspended, respectively, in distilled water (lane2); in 0.0007% Tween-20 (lane 3); in 0.04% sarcosyl (lane 4); and in 0.02% SDS (lane 5). After 10 min of gentle stirring, cells were removed by centrifugation and the presence of PorZ in the cell pellet (lane 1) and supernatants (lanes 2–5 was checked). The detergent concentrations correspond to one-tenth of the critical micelle concentration (CMC). Lane M, MagicMark™ XP Western Protein Standard.