Characterization of αArp2 antibodies.

(A) Affinity-purified αArp2 is specific for rArp2 in bacterial homogenates. Homogenates of IPTG-induced (I, lanes 1 and 3) or uninduced (U, lanes 2 and 4) M15 bacteria carrying the pQE30/Arp2 plasmid were electrophoresed in parallel. Gels were stained with Coomassie blue (lanes 1 and 2) or were Western blotted and probed with affinity-purified αArp2 antibodies (lanes 3 and 4). Coomassie blue–staining of purified recombinant Arp2 demonstrated a single band (lane 5) that was recognized by the affinity-purified antibody in Western blot (lane 6). In this case, 0.5 μg rArp2 has been loaded on the gel. (B) Detection of Arp2 in platelets by Western blot analysis. Platelet proteins were analyzed by Coomassie gels (lane 1) and for the presence of αArp2 in Western blots (lane 2). (C) Detection of native Arp2 in platelet extracts. Platelet extracts were incubated with protein A beads conjugated to either αArp2 (Arp2) or preimmune (Pre) antibodies from the same rabbit as αArp2, and the beads were collected by centrifugation. Supernatants (Sup) and pellets (Pel) were probed by Western blot for Arp2 (αArp2; top panel), p34, another Arp2/3 subunit (αp34; middle panel) or actin (αactin; lower panel). Note that Arp2 is removed from the extract and appears in the pellet after treatment with αArp2 beads but not with preimmune antibodies. Neither p34 nor actin sediments with the αArp2 beads.