The C-terminal domain of EB1 heterodimerizes with the full-length EBs. (A) Schematic representation of EB constructs used for the heterodimerization and co-IP assays. CHD, calponin homology domain; L, linker; CC, coiled coil; Ac, acidic tail. (B and C) Analysis of the indicated proteins or protein mixtures by native 15% (B) or 9% (C) PAGE at 4°C. EBc fragments were mixed in an equimolar ratio (B); EB1c and the full-length EB1 and EB3 were mixed in different ratios as indicated (C). Heterodimers are indicated by arrowheads. (D) Analysis of the indicated proteins by denaturing SDS-PAGE; molecular weight markers are indicated on the right. (E) Co-IPs of HA-tagged EB1 mutants expressed in COS-1 with an HA antibody. Western blots were probed with the indicated antibodies. EB1-C and EB1-CΔAc but not EB1-NL co-precipitate the endogenous EBs. Extr. = 10% of the cell extract, used for IP. (F) Co-IPs of endogenous EBs from COS-1 cells. Extr. = 10% of the cell extract, used for IP.