Figure 2: ARPC1B and Arp2/3 complex are deficient in ARPC1B mutant platelets.

(a) Crystal structure of mammalian Arp2/3 showing relative location of component proteins from the protein data bank (PDB1K8K, http://www.rcsb.org/pdb/explore/explore.do?structureId=1K8K; Robinson et al.¹⁶). (b) Immunoblot analysis of platelet lysates showed ARPC1B to be absent in Patient 1 (P1) and greatly reduced in Patients 2 and 3 (P2, P3) compared to normal (N), while levels of ARPC2, ARPC3, ARPC5, ARP2 and ARP3 were normal (gamma tubulin used as loading control, see Supplementary Fig. 3a). (c) Platelet ARPC1A was increased in all three patients relative to normal, while WASP expression was equivalent (GAPDH used as loading control). (d) Immunoblot analysis of a normal platelet lysate after native gel electrophoresis showed a band corresponding to Arp2/3 complex detected by probing for ARPC1B (shown) or other Arp2/3 components (Supplementary Fig. 3d). This band was resolved on a second dimension SDS–PAGE gel, and immunoblotting confirmed the presence of Arp2/3 components (ARPC1B, ARPC2, ARP2 and ARP3 shown). (e) Immunoblotting of platelet lysates after native gel electrophoresis for ARPC5 (left) showed a greatly reduced level of Arp2/3 in Patient 1 (P1) platelets relative to normal (N). ARPC1A (right) was detected in the Arp2/3 complex in Patient 1 (P1) but not in normal (N) platelet lysate (native GAPDH tetramer used as loading control).