Fig. 5. Inhibition of immune responses by Nef. (A,B) Induction of Dpt mRNA by bacterial infection was determined by northern-blot analysis (top). 18S rRNA was used as a loading control (bottom). (C) Intracellular localization of Relish was determined by immunostaining with anti-Relish antibody (left). BOBO-3 iodide was used to visualize the nuclear structure (middle). Merged images (right) are shown. (D) Induction of Dpt mRNA by DTRAF2 overexpression was determined by northern-blot analysis (top). 18S rRNA was used as a loading control (bottom). (E,F) Induction of Dpt mRNA by bacterial infection (E) or DTRAF2 overexpression (F) was determined by northern-blot analysis (top). 18S rRNA was used as a loading control (bottom). Fly genotypes were as follows. C: Control, hsp70-GAL4/+. nef or 1xnef: hsp70-GAL4/+; UAS-nef/+. 2xnef: hsp70-GAL4/+; UAS-nef/UAS-nef. G2A: hsp70-GAL4/UAS-nef-G2A. nef+JNK^DN: UAS-JNK^DN/+; hsp70-GAL4/+; UAS-nef/+. nef+hep¹: hep¹/hep¹; hsp70-GAL4/+; UAS-nef/+. T: EP(X)1516/X; hsp70-GAL4/+. T+N: EP(X)1516/X; hsp70-GAL4/+; UAS-nef/+. T+N+JNK^DN: EP(X)1516/UAS-JNK^DN; hsp70-GAL4/+; UAS-nef/+. To induce ectopic gene expression, third-instar larvae were heat shocked for 30 minutes at 37°C and subsequently incubated for 90 minutes at 25°C [A,B,D (left three lanes),E] or heat shocked for 2 hours at 37°C [C,D (right three lanes),F].