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Fig. S1. Conjugation of VB-201 and VB-207 to ovalbumin (OVA) and biotin. (a) Enzyme-linked immunosorbent assay (ELISA) plates were coated with 1 μg OVA, OVA-VB201 or OVA-VB207. Serum from KLH-VB201-immunized mice was diluted as indicated and applied to coated plates. Binding was detected using horseradish peroxidase (HRP) anti-rabbit antibody. Binding levels of OVA to VB-201 and VB-207 were comparable. (b) Precipitation of samples from THP-1 cells with OB-VB201 and OB-VB207 is described in Materials and methods. Whereas comparable amounts of BO-VB201 and BO-VB207 were used for precipitation, Toll-like receptor (TLR)-2 was pulled down only with BO-VB201.

Fig. S2. Generation and enrichment of bone marrow-derived dendritic cells (BMDC). Double staining for CD11c and major histocompatibility complex (MHC) class II of (a) freshly isolated bone marrow, (b) bone marrow cells cultured for 5 days with granulocyte–macrophage colony-stimulating factor (GM-CSF) and (C) CD11c-enriched bone marrow cells cultured for 5 days with GM-CSF.

Fig. S3. VB-201 associates to the membrane fraction of cells. U-937 cells were incubated for the indicated time-points with [3H]2-VB-201. Cells were then collected, centrifuged for 3 min at 5800 g and washed three times with phosphate-buffered saline (PBS); 0·5 ml hypotonic buffer was added to the cell pack for 10 min on ice. Lysates were spun for 5 min at 1100 g. To obtain the cytosolic and membrane fraction, supernatant was collected and spun again at 7700 g. For the membrane fraction, supernatant was loaded onto ultracentrifuge tubes and centrifuged at 19.2 K g for 30 min at 4°C. Results are mean ± standard deviation of triplicates. One of two experiments is shown.

Fig. S4. VB-201 inhibits Toll-like receptor (TLR)-2- and TLR-4-induced phosphorylation in mouse peritoneal macrophages. Thioglycolate-elicited mouse peritoneal macrophages were pretreated at the indicated VB-201 concentrations and then activated with TLR-2 and TLR-4 agonists. Samples were analysed by Western blotting for inhibition of phosphorylation. α-Tubulin was used for loading control.

Fig. S5. VB-201 does not inhibit Toll-like receptor (TLR)-5, -7 and -9 and interleukin (IL)-1 receptor-mediated phosphorylation. Mouse bone marrow-derived dendritic cells (BMDC) (a) and human monocytes (b) (106/ml) were pretreated for 20 min with VB-201 followed by 15 min activation with 10 μg/ml cytosine–phosphate–guanosine (CpG) (TLR-9) oligodeoxynucleotide (ODN) 1826 (mouse) or CpG ODN 2006 (human), 0·5 μg/ml R848 (TLR-7,-8), 1 μg/ml flagellin (TLR-5) or 200 ng/ml recombinant mouse/human interleukin (IL)-1. Samples were analysed by Western blotting for inhibition of phosphorylation. α-Tubulin was used for loading control. One of at least three experiments is shown.

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