Figure 5

LPI effects on Stx binding are independent of signalling and ATP.

(a) HEp-2 cells were pretreated with LPI (10 μM) or GPR55 agonist O-1602 (10 μM) for 10 min prior to addition of Stx1m (100 ng/ml) for 30 min at 37 °C. Cells were washed, fixed, permeabilized and blocked in 5% FBS for 30 min prior to incubation with primary and secondary antibodies. Confocal microscopy images show Stx staining in white and nuclei in blue; scale bar 10 μm. (b) Cells were pretreated with or without 2-deoxy-D-glucose (50 mM) and NaN₃ (10 mM) for 1 h to deplete cellular ATP before 10 μM LPI was added and incubation was continued for 30 min. Cells were then cooled down for 10 min and ¹²⁵I-Stx1m was added for 20 min on ice. Cells were washed, lysed and the radioactivity signal was counted. Results are presented as % of control (mean ± STD; n = 3 for ctrl and n = 2 for LPI). (c) Cells were washed with HEPES-buffered medium to remove serum proteins and fixed with 10% formalin solution for 30 min. The fixed cells were treated with 10 μM LPI for 30 min at 37 °C and then incubated with ¹²⁵I-Stx1m for 20 min at 37 °C. Cell-associated ¹²⁵I-Stx1m was counted and presented as % of control (mean ± SEM; n = 3).