A. phagocytophilum infection induces IPAK1 and IPI3K activity through Gβγ stimulation but independent of Rac1/Cdc42 activation. (A) Lysates from ticks infected (I) or not (UI) with A. phagocytophilum were immunoprecipitated (IP) with anti-PAK1, and PAK1-mediated phosphorylation of the substrate MBP was analyzed by in vitro kinase assay. Total lysates used for the kinase assay were probed with actin antibody as the loading control (input). (B) IPI3K activity read out by ELISA detecting PI conversion to PI(3)P, IPI-3 immunoprecipitates from lysates of A. phagocytophilum–infected or uninfected ticks. (C and D) IPAK1 activity in ipi3k-silenced ticks (C) or igβ- or igγ-silenced ticks (D) in comparison with their respective mock controls was measured as in A. Total lysates used for the kinase assays were probed with actin antibody as the loading control. In A, C, and D, IPAK immunoprecipitates were used at three different dilutions indicated by wedges (10, 15, and 25 µl IP beads). (E) IPI3K activity in igβ- or igγ-silenced ticks in comparison with the mock control was determined as in B. (F) Rac1/Cdc42 activation upon binding to PAK-PBD (Rac1/CDC42 binding domain of PAK1) upon A. phagocytophilum infection. Total tick lysates were used for affinity precipitation of Rac1/Cdc42 GTPases with PAK-PBD beads. Total lysates before precipitation were probed with actin as the loading control. Statistics were performed using the Student’s t test, and the p-value is shown in B and E. Error bars show mean + SD. Representative data from two independent experiments is shown in all panels.