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. 2009 Mar 27;284(13):8984–8994. doi: 10.1074/jbc.M809719200

FIGURE 6.

FIGURE 6.

Laminin and type IV collagen assembly on cell surfaces. Schwann cells were incubated with the indicated components (14 nm each unless otherwise indicated1) for 1 h, washed, fixed, and immunostained for laminin, agrin, nidogen/entactin, and/or collagen IV and counterstained with 4′,6-diamidino-2-phenylindole (blue). Panels A, B, E, F, I, and J. Cells were untreated (NT), treated with (wt) laminin, LmΔαLN-L4b, LmΔαLN-L4b + αLNNd, LmΔαLN, and LmΔαLN + αLNNd and immunostained with anti-Lmγ1 (green) antibody. Increased laminin immunofluorescence was detected when αLNNd was coincubated with laminins lacking the αLN domain. Panels M and N, αLNNd was not detected on cells when incubated alone (anti-entactin) but was detected colocalized with laminin when co-incubated with LmΔαLN. Panels C, D, G, and H, cells were treated with LmΔLG, LmΔLG + mAgrin (mA) and stained with antibodies for laminin (anti-Lmγ1, green) or agrin (red). Increased laminin and mA immunofluorescence was detected when mA was incubated with LmΔLG. Panels K and L, cells were incubated with LmΔ(αLN&LG) + mA or with LmΔ(αLN&LG) + mA + αLNNd and stained with antibody for Lm. Increased laminin immunofluorescence was detected when LmΔ(αLN&LG) was co-incubated with mA and αLNNd but not when incubated only with mA. Panels M, and N, αLNNd (M; 14 nm) incubated alone (M, anti-entactin, red; anti-Lmγ1, green) or with LmΔαLN (N; 14 nm). Little αLNNd accumulated on cell surface in the absence of the laminin. Panels O and P, when Myc-tagged laminin (56 nm) was incubated, both Myc and LG4-5 (E3) epitopes were detected. When Myc-tagged LmΔLG (28 nm) was incubated with Myc-free laminin (28 nm), only the LG4-5 epitope (compared with Myc) was detected on the cell surface. Panels Q-V, cells were immunostained for collagen IV (red) after treatment with LmΔαLN-L4b + collagen IV (Col-IV), laminin + Col-IV, LmΔαLN-L4b + Col-IV + nidogen (Nd), laminin + Col-IV + Nd, LmΔαLN-L4b + Col-IV + αLNNd, and laminin + Col-IV + αLNNd. Increased collagen IV was detected with either αLNNd with non-polymerizing laminin or nidogen with either wt or non-polymerizing laminin.