Myo10 directly interacts with Wee1 via the MyTH4 domain. (A) In vitro binding assay between recombinant FLAG-Wee1A and empty glutathione-sepharose beads (blank) or bead-bound GST or GST-MyTH4-FERM (GST-4F). Inputs and washed glutathione pellets were boiled in SDS sample buffer and immunoblotted for GST and Wee1 by standard methods. (B) Bacterial recombinant sepharose-bound GST and GST-MyTH4 (GST-4) were mixed with concentrated extracts from stage 10 X. laevis embryos. Washed pellets (pel) and raw extract (input) were boiled in SDS sample buffer and subjected to immunoblot analysis. Input sample represents 2% of assay input and pellets each represent half of the pull-down product. Membranes were cut at the 50-kD marker and immunoblotted for Wee1 (top) or GST (bottom). The lower GST-reactive bands in the GST-4 pel sample represent protein degradation products. (C) Same as B, except using recombinant GST-MyTH4-DD (GST-4-DD) protein. Numbers on the left side of the blots in A–C indicate molecular weight in kilodaltons. (D) Stage 10 embryos were prepermeabilized before fixation and immunostained with antibodies against α-tubulin (microtubule [MT] not in merge), Wee1B (green), and Myo10 (red). The Myo10 antibody is conjugated with Alexa Fluor 568 and was applied after the anti–rabbit secondary used to label the Wee1B antibody. Bar, 10 µm.