Fig. 1. Identification of human cellular proteins associated with the cofilin-actin complex. (A) HEK293 cells were transfected with either a plasmid that drove the expression of Cof-His₆ (lane +), or a control vector (lane -). Cell lysate (3 mg protein equivalent) was incubated with Ni²⁺-NTA resin to adsorb Cof-His₆ and associated proteins. Bound material was eluted with step-wise increments in the concentration of NaCl. The eluate was electrophoresed on a 10-20% polyacrylamide gradient gel and silver stained. The lanes of the 300 mM NaCl eluates ares shown. The arrowheads point to bands that were recovered specifically from Cof-His₆-producing cells. A small amount of Cof-His₆ leaked out of the resin as a result of NaCl washing (arrow). (B) HA-tagged actin-binding proteins (p57^WD, fascin, CAP1 and EF1) were expressed separately in HEK293 cells with (+) or without (-) co-expression of Cof-His₆. Cof-His₆-associated protein fractions were prepared as above. Crude lysates (lysate) and 500 mM NaCl eluates (bound) were subjected to western blotting with an anti-HA MAb (clone 12CA5). The size of the bands is around 55kDa. (C) Anti-CAP1 blotting of p55. Crude lysate (lane 1) and 300 mM NaCl eluates from Ni²⁺-resin (lanes 2 and 3) were subjected to western blotting with an affinity-purified rat IgG to CAP1. The NaCl eluate from Cof-His₆-producing cells is clearly positive for the CAP1 signal (lane 3), in contrast to the eluate from non-expressing cells (lane 2).