Western blotting analyses. Validation of Amblyomma americanum AamAV422 protein secretion into the host, cross-reactivity of recombinant (r)AamAV422 with antibodies to other ticks, temporal and spatial expression analyses of AamAV422 were done by routine western blotting analyses as described in section 2.6. rAamAV422 western blots were screened with rabbit pre-immune serum (A), antibodies to 24 h (B) and 48 h (C) A. americanum tick saliva proteins (TSPs) and antibodies to Ixodes scapularis (D) TSPs. Mono-specific antibodies were eluted as described and used to screen western blots of tick saliva (TS) proteins, tick hemolymph (TH) and protein extracts of whole ticks (WT) (E), and dissected tick organs, salivary glands (SG), Midgut (MG) and carcass (CA) (F). TS lanes 1, 2 and 3 are TSPs harvested from 48, 96 and 120 h fed ticks, respectively. TH, WT, SG, MG and CA lanes 1, 2 and 3 are hemolymph and protein extracts of 24, 72 and 120 h fed ticks. PC, rAamAV422 used as positive control; *, a potentially glycosylated native AamAV422 form.