Figure 3.
Roles of individual subunits in maintaining complex integrity. (A) Characterization of anti-Arp3p and anti-Arc40p antibodies. a and b are immunoblots of extracts prepared from a wild-type strain (RLY1) and a strain expressing (myc)5-(his)6-tagged Arp3p as its sole copy of Arp3p (RLY188), respectively, by using affinity-purified anti-Arp3p antibody. c and d are immunoblots of extracts prepared from a wild-type strain (RLY1) and a strain expressing both wild-type and green fluorescent protein-tagged Arc40p (RLY821), respectively, by using affinity-purified anti-Arc40p antibody. (B) Extracts from wild-type and subunit-deletion strains were prepared as described in Materials and Methods and run on an S1000 gel filtration column at a flow rate of 0.5 ml/min. The fractions were trichloroacetic acid-precipitated and analyzed by using immunoblotting with antibodies against Arp2p (16), Arp3p, and Arc40p. (C) Subunit composition of Arp3p-associated complexes purified from the wild-type (RLY188) and Δarc15 (RLY763) strains as described in Materials and Methods. Complexes were run on SDS/15% polyacrylamide gels and visualized by using Coomassie blue staining.