Adult female mosquitoes were injected each with 69 nl (approx. 4 × 105 bacteria) of a 20 mg/ml suspension of pHrodo-labeled E. coli bioparticles (Invitrogen) in sterile PBS, and hemolymph was collected directly into nonreducing SDS-PAGE sample buffer from groups of 30 mosquitoes at 1, 3 and 12 h after bioparticle injection. Hemolymph proteins were separated by SDS-PAGE and transferred to Immunoblot polyvinylidene fluoride membrane (BioRad) using wet transfer (BioRad). Blots were incubated with mouse anti-CLIPA2, rabbit anti-TEP1 [25 (link)] and rabbit anti-PPO6 [26 (link)] polyclonal antibodies at dilutions of 1/1,000, 1/1,000 and 1/2,000, respectively. Anti-mouse and anti-rabbit IgG horseradish peroxidase-conjugated secondary antibodies (Promega) were used at 1/6,000 and 1/12,000, respectively.
A bioparticle surface extraction assay was performed exactly as previously described [12 (link)]. Proteins in the soluble and bound fractions were resolved by nonreducing SDS-PAGE, and Western blot was performed as described above.
To study the effects of TEP1 and LRIM1 kd on CLIPA2 protein levels in the absence of infection, hemolymph was extracted from naive mosquitoes 48 h after dsRNA injection. Hemolymph proteins were resolved by nonreducing SDS-PAGE, and Western blot was performed as described above. Rabbit anti-LRIM1 polyclonal antibody was used at 1/2,000 [20 (link)].
A bioparticle surface extraction assay was performed exactly as previously described [12 (link)]. Proteins in the soluble and bound fractions were resolved by nonreducing SDS-PAGE, and Western blot was performed as described above.
To study the effects of TEP1 and LRIM1 kd on CLIPA2 protein levels in the absence of infection, hemolymph was extracted from naive mosquitoes 48 h after dsRNA injection. Hemolymph proteins were resolved by nonreducing SDS-PAGE, and Western blot was performed as described above. Rabbit anti-LRIM1 polyclonal antibody was used at 1/2,000 [20 (link)].
