Additional file 2: of A novel TLR4 binding protein, 40S ribosomal protein S3, has potential utility as an adjuvant in a dendritic cell-based vaccine

Figure S1. NF-κB activity was measured using cancer cell lysates to assess TLR4-expressing tumor cells. Figure S2. To identify the TLR4 binding proteins, two groups were divided and assessed through pull-down assay. Figure S3 Mouse BMDCs were treated with doxorubicin (0, 0.1, 1, 10 μg/mL). Figure S4. For endotoxin contamination test, recombinant RPS3 (1 μg) and LPS (100 ng) were incubated with proteinase K (100 μg/ml) or with polymyxin B (10 μg/ml). Figure S5 Mouse BMDCs were separated into RPS3 treated mature DCs loaded with OVA peptide or E7 peptide. Figure S6. To confirm the dominancy of T cells in adaptive immune responses to clear tumors, T cell or NK cell depletion antibodies were injected into mice before the injection of cancer cells. Figure S7 We examined the TLR4-dependency for DC activation and maturation by using TLR4 blocking antibody. Figure S8 The purification of recombinant HMGB1 protein was confirmed by CBB staining and by western blot (A). Figure S9 HEK293 cells were transfected by shRNA (GFP) as a negative control or shRNA (RPS3). Figure S10 Mice serum with vaccination or not were used to confirm that RPS3 does not induce humoral immunity, producing autoantibodies against itself. (DOCX 1509 kb)