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Fig. 8. Transient phosphorylation of ezrin in ACHN cells after treatment with the Stx1-B subunit. (A) Total cell extracts were prepared from ACHN cells treated with or without 5 µg/ml of Stx1-B subunit for the indicated periods. After separation on 10% SDS-PAGE gel, the proteins were transferred to a nitrocellulose membrane and immunoblotted using the indicated antibodies. (B) Total cell extracts were prepared from ACHN cells treated with or without different amounts of Stx1-B subunit for 10 minutes and immunoblot analysis was performed using anti-phospho-specific ezrin antibody (P-Ezrin) as in A. Intensity of the phospho-ezrin signals obtained from each sample was quantitated by densitometry and the relative value of each to that of untreated cells (each value/the value of untreated cells) was indicated as a graph (lower panel). (C) Total cell extracts were prepared from ACHN cells treated and not treated with 5 µg/ml each of either anti-globotriaosyl ceramide antibodies (38.13, 1A4) or Stx1-B subunit for 10 minutes and examined by immunoblotting as in A. (D) Five µg/ml of Stx1-B pentamer was bound to the cell surface of the ACHN cells by incubation for 30 minutes at 4°C. After intensive washing with ice-cold PBS to remove the excess toxin, temperature was shifted from 4°C to 37°C to allow intracellular stimulation and cells were incubated for the time periods indicated in the figures. Immunoblot analysis was performed as in A.
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