Fig. 4
AGSLs of the cell membrane were essential for the actions of βγ-CAT. a THP-1 cells were treated with PPMP and then incubated with FITC-labeled βγ-CAT. The binding between βγ-CAT and THP-1 cells was detected by flow cytometry. b The oligomerization of βγ-CAT was determined by western blotting. The immunoblots are representative of three independent experiments, the original images of immunoblots are shown in Supplementary Figure 12. c The IL-1β release induced by βγ-CAT was measured by ELISA. *P < 0.05 vs. control by using unpaired two-tailed Student’s t test (n = 3). d–f THP-1 cells in which ganglioside expression was knocked down or gangliosides re-addition, then incubated with βγ-CAT. The binding between βγ-CAT and THP-1 cells was detected by flow cytometry (d), the oligomerization of βγ-CAT was detected by western blotting (e), and the IL-1β concentration was measured by ELISA (f). *P < 0.05 vs. the respective control by using unpaired two-tailed Student’s t test (n = 3). The immunoblots (e) are representative of three independent experiments, the original images of immunoblots are shown in Supplementary Figure 13. g–i THP-1 cells in which sulfatide expression was knocked down or sulfatides re-addition, then incubated with βγ-CAT. The binding between βγ-CAT and THP-1 cells was detected by flow cytometry (g), the oligomerization of βγ-CAT was detected by western blotting (h), and the IL-1β concentration was measured by ELISA (i). *P < 0.05 vs. the respective control by using unpaired two-tailed Student’s t test (n = 3). The immunoblots (h) are representative of three independent experiments, the original images of immunoblots are shown in Supplementary Figure 13. In flow cytometry (a, d, g), the untreated THP-1 cells are indicated as normal, and THP-1 cells treated with FITC-labeled βγ-CAT are indicated as control. Bars represent the mean ± SD from three independent experiments per condition in c, f, i
