Fig. 3.

Syk activation is required for the induced dissociation of cPLA₂α–AnxA1. a HeLa cells were transfected with an Syk-expressing plasmid for 24 h before being treated with 1 µg/ml ShigaB-sulf2 (StxB-S2) or cholera toxin (Ctx) for the indicated times, or 1 µM H₂O₂ for 10 min, and the protein lysates were subjected to immunoblot analysis using antibodies against phoshorylated Syk (pSyk), total Syk (totSyk) and GAPDH. b Quantification of immunoblots as shown in a, with pSyk levels normalized to GAPDH and presented as % of control (n = 4 independent experiments, mean values +SEM, *p < 0.05). HeLa cells were pretreated with the indicated inhibitors for 30 min and treated with either 1 µg/ml StxB-S2 (c) or Ctx (d) for 10 min before being fixed, permeabilized and subjected to PLA using cPLA₂α and AnxA1 antibodies. Images were taken by an LSM780 confocal microscope, and the number of signals per cell was quantified and presented as % of control. The mean values of at least three independent experiments are shown as +SEM, *p < 0.05. e HeLa cells stably expressing GFP-Syk were transfected with either control siRNA or Syk-specific siRNAs, and after 3 days the protein lysates were subjected to immunoblot analysis using antibodies against Syk and GAPDH. f HeLa cells transfected as described in e were treated with 1 µg/ml StxB-S2 or Ctx for 10 min before being fixed, permeabilized and subjected to PLA as described in c, d (n = 3 independent experiments, mean values +SEM, *p < 0.05)