Fig. 5.

SDS/PAGE analysis of azidoblebbistatin crosslinking of Dd proteins. Azidoblebbistatin-attached proteins in the myosin-enriched fraction of DdMd-expressing Dd cell lysates were detected by fluorescence of the covalently bound inhibitor (A) and, on the same gel, the protein contents were analyzed by subsequent Coomassie staining (B). Purified DdMd was loaded in the right-most lane of the gel as a control. Fluorescent bands are indicated at the left side of A in the order of increasing mobility. (C–H) Azidoblebbistatin concentration dependence of the fluorescence intensity of azidoblebbistatin-crosslinked protein bands. Hyperbolic fits to the datasets indicated high-affinity azidoblebbistatin binding in the case of bands in which the myosin II motor domain was present (C–E) and low-affinity binding in the case of other target proteins (F and G). Determined EC₅₀ values are shown in Table 1.