A Quantitative RNAi Screen for JNK Modifiers Identifies Pvr as a Novel Regulator of Drosophila Immune Signaling (original) (raw)
Figure 3
Pvr is a negative regulator of dJNK phosphorylation in the IMD pathway.
(A,B) Quantification of PGN-induced dJNK phosphorylation relative to f-actin (A) or total JNK (B). S2 cells were incubated with the indicated dsRNAs and exposed to PGN at 0min, 15min or 60min as indicated. Key and dTak1 dsRNA were used as modifier dsRNA controls, whereas Toll and CG11318 dsRNA were used as non-modifier dsRNA controls. Cells were stained with anti-P-JNK antibody and dJNK phosphorylation was standardized to f-actin (A) and total dJNK (B). Data are presented as the mean of two independent experimental values and error bars indicate + SEM. The grey dashed line represents the mean dJNK phosphorylation value for Toll dsRNA and dsRNAs that significantly modulated dJNK phosphorylation from this value are indicated (* = p-value <0.05, ** = p-value <0.01). Secondary dsRNA analysis recapitulates the dJNK phosphorylation values from the primary screen. (C) A partial genetic and physical interaction network of dJNK phosphorylation modifiers. Modulators of dJNK phosphorylation with z-scores greater than 1.96 or less than −1.96 were grouped in an interaction network using known genetic and physical interaction databases. Within this network, Pvr (black circle) and dJnk (white circle) are connected directly and through a number of intermediate genes (yellow circles). The Pvr and dJnk interaction network also connects to IMD pathway (blue circles). (D) Quantitative Western blot analysis of lysates from S2 cells treated with either Pvr or GFP dsRNA. Lysates were probed with anti-Pvr (top blot) and anti-actin (bottom blot) antibodies. Pvr levels were quantified relative to actin (top graph). Data are presented as mean of three independent experiments and error bars indicate + SEM. Both Pvr dsRNA molecules deplete Pvr in S2 cells. (E) Quantification of PGN-induced dJNK phosphorylation. S2 cells were treated with GFP dsRNA as a control or two independent non-overlapping dsRNA targeting Pvr as indicated. Cells were exposed to PGN and dJNK phosphorylation was monitored relative to total dJNK. P-JNK:JNK values at 0h PGN exposure with GFP dsRNA were assigned a value of 1 and the remaining P-JNK:JNK values are reported relative to these data. Data is expressed as the mean of two independent experiments and the error bars represent +/− SEM. Significant differences in pJNK values are indicated (* = p-value<0.05, ** = p-value<0.01). Depletion of Pvr increases PGN-induced dJNK phosphorylation at 15min, indicating that Pvr negatively regulates dJNK activation in the IMD pathway.