Figure 6

Characterization of LPI-induced inhibition of Stx binding to Gb3.

(a) HEp-2 cells were pretreated with increasing concentrations of LPI for 30 min at 37 °C or on ice, prior to cooling down and incubation with ¹²⁵I-Stx1m for 30 min on ice. Cells were washed, lysed and the radioactive signal was measured. Results are presented as % of control (mean ± SEM; n = 3). (b) Cells were pretreated with increasing concentrations of LPI for 30 min at 37 °C, prior to incubation with ¹²⁵I-Stx1m for 20 min at 37 °C. Cells were washed, lysed and the radioactive signal was measured, results are presented as % of control (mean ± SEM; n = 3). (c) Cells were pretreated with 10 μM LPI for 30 min in absence or presence of 10% FBS prior to incubation with ¹²⁵I-Stx1m for 20 min at 37 °C. The quantification of radioactivity measurements is shown, presented as % of control (mean ± SEM; n = 3). (d) Cells were incubated with 10 μM LPI for 30 min and then washed once at 37 °C with HEPES-buffered cell growth medium with or without 10% FBS, prior to cooling down on ice for 10 min and incubation with ¹²⁵I-Stx1m on ice for 20 min. The quantification of radioactivity measurements is shown, presented as % of control (mean ± STD; n = 2). (e) Cells were pretreated with 10 μM LPI for 30 min prior to addition of Stx1m (100 ng/ml) or anti-Gb3 antibody and incubated for 20 min at 37 °C. Cells were washed, fixed, permeabilized and blocked in 5% FBS for 1 hr prior to incubation with primary (Stx samples only) and secondary antibodies. Confocal microscopy images show Stx or anti-Gb3 antibody staining in white and DAPI nuclear staining in blue; scale bar 10 μm. (f) Quantification of intensity signals of Stx and anti-Gb3 antibodies from confocal images, expressed as % of control (mean ± SEM; n = 3).