Fig. 2
The BmALP1 and BmTFF3 subunits of βγ-CAT bound to gangliosides and sulfatides, respectively. a Schematic graph of βγ-CAT, BmALP1 subunit and BmTFF3 subunit. The BmALP1 subunit and BmTFF3 subunit of βγ-CAT have apparent molecular weights of 38 kDa and 18 kDa, respectively. b Interactions between MBP-αN and MBP-αC with gangliosides was detected by BLI assay. c Interactions between MBP-αN and MBP-αC with sulfatides was detected by BLI assay. d The binding kinetic curves between MBP-αC and gangliosides were determined by BLI assay. e βγ-CAT was incubated with different concentrations of anti-BmTFF3 polyclonal antibody or control IgG and added to LPS-primed THP-1 cells. The IL-1β concentration in the supernatant was measured by ELISA. Bars represent the mean ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. control IgG by using unpaired two-tailed Student’s t test (n = 3). f FITC-labeled βγ-CAT was incubated with an anti-BmTFF3 polyclonal antibody or control rabbit IgG and added to THP-1 cells, the binding of βγ-CAT with THP-1 cells was evaluated by flow cytometry. Untreated THP-1 cells are indicated as normal, and THP-1 cells treated with FITC-labeled βγ-CAT are indicated as the control. g βγ-CAT was incubated with an anti-BmTFF3 polyclonal antibody or control rabbit IgG and then added to THP-1 cells, the oligomerization of βγ-CAT was detected by western blotting. The immunoblots are representative of three independent experiments, the original images of immunoblots are shown in Supplementary Figure 8. h The binding of BmTFF3 with different types of sphingolipids was determined by protein-lipid overlay assay. The blots are representative of three independent experiments, the original images of blots are shown in Supplementary Figure 8. i The binding kinetic curves between BmTFF3 and sulfatides was determined by BLI assay. The BLI interaction curves in b–d and i are representative of three independent experiments
