Fig 4. Serpin-5 and serpin-9 inhibit melanization in vitro.
(a) The relative expression levels of serpin-5 and serpin-9 were validated by qPCR analysis at 24, 48 and 72 hpi. Data are present at least three independent experiments (mean and s.e.m). Statistical significance was determined using a two-tailed Student’s t-tests.* p ≤ 0.05, ** p ≤ 0.01. Sample abbreviations: MF, fat body from mock group; IF, fat body form infection group; MH, hemocytes from mock group; IF, hemocytes form infection group. (b) Purification of recombinant serpin-5 and serpin-9 proteins from E. coli. Purified proteins were resolved by SDS-PAGE. (c) Concentration-dependent inhibition of spontaneous melanization by serpin-5 and serpin-9. 1μL hemolymph from naïve 5th instar larvae mixed with different amounts of serpin-5 (0–320 μg/mL) or serpin-9 (0–1200 μg/mL) was incubated at room temperature for 5 min before measuring PO activity. A recombinant baculovirus protein, P33, was used as negative control.