Skip to main content
. 2008 Dec 15;183(6):989–997. doi: 10.1083/jcb.200805041

Figure 1.

Figure 1.

α–E-catenin binds to dynamitin and interacts with the dynactin protein complex. (A) Schematic of α–E-catenin containing three vinculin homology (VH) domains. Numbers indicate corresponding amino acids. (B) Interaction between dynamitin and α–E-catenin in a yeast two-hybrid assay. Cells were expressing plasmids containing Gal4 DNA–BD linked to various fragments of α–E-catenin and Gal4-AD linked to dynamitin (Dyn) or β-catenin (β-cat; positive control). (C) Interaction between dynamitin and α–E-catenin in mammalian cells. HEK 293FT cells were transfected with plasmids encoding GST and GST-linked α–E-catenin or vinculin and V5-tagged dynamitin, β-catenin (positive control), and Tbr1 (negative control). Protein extracts were pulled down (IP) with glutathione–Sepharose beads and analyzed by Western blotting (WB) with anti-V5 or anti-GST antibodies. (D) α–E-catenin associates not only with dynamitin but also with Arp1 and p150Glued proteins. GST-tagged full-length or VH2–VH3 fragments (α-cat 291–906) of α–E-catenin, V5-tagged dynamitin, and Arp1 were produced in HEK 293FT cells. GST-fused proteins were pulled down with glutathione–Sepharose beads, and protein complexes were analyzed by blotting with anti-V5, anti-p150Glued, and anti-GST antibodies. (E) α–E-catenin partially cofractionates with dynactin. Total keratinocyte extracts were sedimented on sucrose gradient, and fractions were analyzed by blotting with anti-p150Glued, anti–dynein intermediate chain (DIC), antidynamitin, anti-Arp1, and anti–α-catenin antibodies. (F) Interaction between endogenous α–E-catenin and dynactin. Dynactin-containing fractions after sucrose sedimentation of total extracts from wild-type (WT) and α–E-catenin−/− (KO) cells were immunoprecipitated with anti–α-catenin (C terminal) or β-galactosidase (β-gal; control) antibodies and analyzed by blotting with anti-p150Glued, antidynamitin (Dyn), anti-Arp1, and anti–α-catenin antibodies. M, position of a molecular weight standard band in the marker lane. (G) Interaction between endogenous α–E-catenin and dynamitin. Total proteins (input) from wild-type and α–E-catenin−/− keratinocytes were immunoprecipitated with anti–α-catenin (IP–α-cat; N terminal) antibodies and analyzed by blotting with antidynamitin and anti–α-catenin antibodies.