Fig 5. TG2 is associated to DS/CS side chains of decorin molecules secreted into the culture medium by human keloid fibroblasts.

(A) Molecular complexes that contained native decorin or decorin treated with chondroitinase ABC (Chase ABC) to eliminate DS/CS chain were immunoprecipitated (IP) with an anti-human decorin core protein antibody (anti-dec) from the equal portions of the conditioned medium (each portion contained 5 μg of GAG) and separated by reducing SDS-PAGE followed by Western blot detection with the anti-dec or an anti-human TG2 serum. Lane 1 –the anti-dec antibody applied as negative control; lane 2 –the core protein of decorin; lane 3 –native decorin; lane 4 –components that were co-immunoprecipitated with the decorin core protein and detected by the anti-TG2 antibody; lane 5 –molecules that were co-immunoprecipitated with native decorin and detected by the anti-TG2 antibody; lane 6 –the cross-reactivity between the antibody that was used for the co-immunoprecipitation (mouse anti-decorin) and the sera were applied for the Western blotting (rabbit anti-human Tg2 and goat anti-rabbit IgG). The migration position of mass markers is shown. (B) The equal portions of the conditioned medium (each containing 5 μg of GAG) from the keloid fibroblasts, which have been cultured in the presence of 50 μM putrescine, were untreated or digested with Chase ABC and subsequently incubated with a human recombinant TG2, as described in Materials and methods. Molecular complexes that contained decorin or its core protein were immunoprecipitated and subjected to Western blot analysis firstly for TG2, and after stripping for decorin. Lane 1 –TG2 that co-immunoprecipitated with the untreated decorin; lane 2 –TG2 that co-immunoprecipitated with Chase ABC treated decorin; lane 3 –decorin untreated with Chase ABC; lane 4 –decorin digested with this enzyme. The migration position of the human recombinant TG2 (hrTG2) is indicated by arrowhead. All the experiments were performed in triplicate.