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. 2008 Oct;17(10):1834–1843. doi: 10.1110/ps.035733.108

Figure 3.

Figure 3.

Time course of SPM-mediated cleavage of purified fusion proteins at different calcium concentrations and temperatures. (A) Purified GST-SPM-His, MalE-SPM-CBD, and FrpCΔRTX, respectively, were incubated in the presence of 2 mM free calcium ions at 37°C and pH 7.4. Aliquots of the processed proteins were withdrawn at the indicated times and separated by SDS-PAGE. The processing was quantified by densitometric analysis of the Coomassie-stained gels. (Inset) An example showing the cleavage of the GST-SPM-His fusion protein over time. GST-SPM-His (B,C) and MalE-SPM-CBD (D,E) were incubated for the indicated times with 2, 5, 10, and 50 mM calcium ions at 23°C (B,D) or 4°C (C,E) and pH 7.4. Aliquots of the processed fusion proteins were separated by SDS-PAGE and stained with Coomassie blue. Extent of cleavage of GST-SPM-His and MalE-SPM-CBD was quantified by densitometric analysis of SDS-PAGE gels. The values represent an average of four independent determinations ± standard deviations.