Comparison of pronase susceptibility of ETX complex formed in MDCK II cells at 4°C or 37°C.
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A) Comparison of pronase digestion of ETX complex in intact MDCK II cells at 4°C or 37°C. MDCK II cells were treated as described above with the exception that, following treatment, some MDCK II cells were lysed and then subjected to pronase digestion. Arrows denote, as specified, migration of the ∼155 kDa ETX complex or the ∼30 kDa ETX monomer. B) Fluorescence quantification of pronase dose effects on digestion of ETX complexes in intact MDCK II cells formed at 4°C vs. 37°C. C) Fluorescence quantification of pronase digestion of ETX complexes, formed at either 37°C or 4°C, in intact MDCK II cells vs. MDCK II cell lysates. This analysis showed that ETX complexes formed at either 4°C or 37°C are extensively digested when present in cell lysates but only the ETX complexes formed at 4°C are pronase-digested when using intact cells. These results suggest that in intact cells the ETX complex formed at 37°C has inserted into the membrane. Results shown are the average of 3 experiments.