Functional Dissection of an Innate Immune Response by a Genome-Wide RNAi Screen (original) (raw)
Figure 5
Dnr1 Protein Levels Are Regulated by Dredd Activity
(A) Amounts of HA-Dnr1 transiently increase in S2 cells treated with LPS. Anti-HA Western blot of lysates from HA-Dnr1-transfected S2 cells that were incubated with LPS for indicated periods.
(B) Anti-HA Western blot of lysates from S2 cells transfected with HA-Dnr1. Coexpression of the caspase inhibitor p35 dramatically inhibits HA-Dnr1 accumulation in the absence (lanes 1 vs. 3) or presence (lanes 2 vs. 4) of LPS. Actin levels are shown as a loading control.
(C) Upper panel shows the percentage of cells with nuclear GFP-Relish after the indicated treatments. The lower panel is an anti-GFP Western blot of lysates from S2 cells treated in the identical manner. z-VAD-FMK prevents nuclear accumulation of GFP-Relish and GFP-Relish processing in response to LPS.
(D) Anti-HA Western blot of lysates from S2 cells transiently transfected with HA-Dnr1. While 2 h incubation with LPS normally leads to a 4-fold increase (quantified by titration) in HA-Dnr1 (lanes 1 vs. 2), incubation with z-VAD-FMK prevents the accumulation (lanes 3 vs. 4).
(E) Anti-HA Western blot of lysates from S2 cells transfected with HA-Dnr1. Cells had been previously incubated with (lanes 3 and 4) or without (lanes 1 and 2) Dredd dsRNA. Results are shown for two independent experiments. Actin levels are shown as a loading control.
(F) Anti-HA Western Blot of lysates from S2 cells transfected with HA-Dnr1 shows that prior RNAi against the caspases Dcp-1, Ice, Nc, and Decay does not substantially affect HA-Dnr1 levels (compare with control without LPS).
(G) The number of Dipt-lacZ-expressing cells after LPS treatment is greatly reduced after Dredd RNAi, while RNAi against Dcp-1, Ice, Nc, or Decay has no effect.