Figure 12.

Effect of Drosophila MICAL forms and NADPH on the actin polymerization state. (a) Pyrene-labelled G-actin (1.1 μM final concentration) was incubated with Drosophila MICAL-MOCH (600 nM) and/or NADPH (100 μM) in G buffer (5 mM Tris-HCl pH 8.0, 0.2 mM CaCl₂, 0.2 mM ATP and 1 mM DTT). Polymerization of actin was induced by adding 5 mM Tris-HCl pH 7.5, 50 mM KCl, 2 mM MgCl₂, 1 mM EGTA, 0.5 mM DTT, and 0.2 mM ATP. Fluorescence intensity was immediately monitored at 407 nm with excitation at 365 nm by a fluorescence spectrophotometer (Spectra max M2; Molecular Devices); (b) Drosophila MICAL-MOCH (Mical redoxCH in the figure), NADPH, and/or hydrogen peroxide were added to pre-polymerized pyrene-labelled F-actin (1.1 μM) and fluorescence intensity was immediately monitored as described above; (c) Pre-polymerized F-actin (18.6 μM) was incubated with Drosophila MICAL-MOCH (2.4 μM final concentration), and/or NADPH (200 μM final concentration) for 30 min at room temperature. Then, the samples were subjected to high-speed centrifugation at 150,000 × g for 1.5 h at 24 °C. Supernatants and resuspended pellets were analysed by SDS-PAGE followed by Coomassie blue staining. Experimental conditions have been derived from the procedures described in the Supplementary information of [53]. Reprinted by permission from Macmillan Publishers Ltd.: [Nature] ([53]), copyright (2010).