Figure 6. Phenoloxidase A3 accumulated in Nopp140-depleted larvae. (A) Whole larval lysates from parental control larvae (Act5C-GAL4/CyO, lane 1; homozygous UAS-C4.2, lane 2) were compared with those from Nopp140-depleted larvae (UAS-C4.2/Act5C-GAL4, lane 3; UAS-C4/Act5C-GAL4, lane 4). UAS-C4 is the original stock containing UAS-C4.2 on the 2nd chromosome and UAS-C3 on the 3rd chromosome. The prominent 70 kDa protein in the lysates of Nopp140-depleted larvae (arrow) was identified by LC-MS/MS as phenoloxidase A3. (B) Hemolymph proteins were resolved on a native polyacrylamide gel and stained with Coomassie Blue. Parental control larvae were homozygous UAS-C4.2 (lane 1) and Act5C/CyO-GAL4 (lane 2). Nopp140-RNAi-expressing UAS-C4.2/Act5C-GAL4 larvae (lane 3) with only a few melanotic masses were compared with similar UAS-C4.2/Act5C-GAL4* larvae (lane 4) that had excess melanotic masses. (C) A native gel similar to that shown in panel B but stained for phenoloxidase activity using tyrosine as substrate. Arrows in B and C mark the position of the most prominent phenoloxidase activity. (D) Melanotic masses were found mostly in the midgut of Act5C > UAS-C4.2 larvae. (E) Melanotic masses also occurred at other sites within Act5C > UAS-C4.2 larvae. This large mass accumulated at the tips of the dorsal arms of the pharyngeal sclerite.