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. 2021 Feb 25;12:593161. doi: 10.3389/fimmu.2021.593161

Figure 1.

Figure 1

Analysis of the mAbs conjugated with antigen. The anti-DEC-205 mAbs and the isotype control were chemically cross-linked to 16E5 and characterized by Western blot and flow cytometry. (A) The conjugates were resolved in an 8% SDS-PAGE under non-reducing conditions and analyzed by Western blot. As control, non-conjugated anti-DEC-205 mAbs were used. The membrane was incubated with a rabbit anti-rat IgG polyclonal Ab, followed by a goat anti-rabbit IgG polyclonal Ab-HRP (left panel). To detect 16E5, a different membrane was incubated with an anti-His mAb, followed by a goat anti-mouse IgG polyclonal Ab-HRP (right panel). Signal was developed by chemiluminescence. (B) BM-derived DCs or MA-104 cells (MA) were lysed and 20 µg of total protein were developed in an 8% SDS-PAGE under non-reducing conditions and analyzed by Western blot. The membrane was incubated with the anti-DEC-205 mAb, followed by a rabbit anti-rat IgG polyclonal Ab-HRP (left panel). A different membrane was incubated with the anti-DEC-205:16E5 or isotype:16E5 conjugates, followed by the anti-His mAb and by a goat anti-mouse IgG polyclonal Ab-HRP (right panel). Signal was developed by chemiluminescence. (C) BM-derived DCs were stained with the anti-DEC-205 mAb, followed by a goat anti-rat IgG polyclonal Ab-FITC, or stained with the anti-DEC-205:16E5 or isotype:16E5 conjugates, followed by the anti-His mAb and a goat anti-mouse IgG polyclonal Ab-FITC. The cells were analyzed by flow cytometry.