FIGURE 8.
HP8 stimulates synthesis of antimicrobial plasma proteins in vivo. Fifth-instar day-0 larvae were injected with buffer (116 μl of buffer 4) or recombinant protein mixtures in 116 μl of buffer 4: HP6Xa (30 ng) activated by bovine Factor Xa (120 ng), proHP8 alone (1.6 μg), or HP8 (1.6 μg) activated by HP6Xa (30 ng). After 20 h, hemolymph was collected, and fat body RNA samples were prepared from each insect. mRNA levels for the indicated genes were assayed by quantitative RT-PCR (Relative Expression) as described under “Experimental Procedures.” A, antimicrobial peptide mRNA expression. B, lysozyme mRNA and plasma lysozyme expression (detected by enzyme assay and immunoblotting (1 μl/lane)). Ab, antibody. C, antimicrobial activity of plasma assayed against E. coli or M. luteus and identification of induced antimicrobial plasma proteins by 15% SDS-PAGE and MALDI-TOF peptide mass fingerprinting. The bars represent mean ± S.D. (n = 3). Bars labeled with different letters (a, b, and c) are significantly different (analysis of variance and Newman-Keuls test, p < 0.05).