MOESM4 of AGI-134: a fully synthetic α-Gal glycolipid that converts tumors into in situ autologous vaccines, induces anti-tumor immunity and is synergistic with an anti-PD-1 antibody in mouse melanoma models

Additional file 4: Figure S4. Binding of mouse anti-Gal antibodies and complement deposition on AGI-134-treated mouse melanoma cells. (A–D) B16-F10 or JB/RH cells were treated with the indicated AGI-134 concentrations or the negative control glycolipid FSL-A. The cells were then incubated with mouse anti-Gal IgM, serum from non-immunized (low anti-Gal titers) or PKH-immunized (high anti-Gal titers) α1,3GT−/− mice, or anti-blood group A,B primary antibodies or buffer only. Anti-Gal antibody binding or deposition of the complement factors C3b/i and C5b-9 were detected using antibodies and flow cytometry. Histogram overlays for the various samples are plotted for representative data from several experiments performed. Of note, the samples for anti-FSL-A and complement deposition in (D) were run in parallel in the same experiment.