Fig. 4.

Quantitative 14-3-3 protein capture identified RAI as a novel 14-3-3 target protein that regulates dsRNA-induced apoptosis and TNF production. A, 646 distinct 14-3-3 target proteins were identified and quantified from HaCaT cell lysate using quantitative 14-3-3 protein capture, and of these 209 proteins had changed affinity to 14-3-3s after dsRNA transfection (left panel). The proteins whose 14-3-3 capture was altered by dsRNA were classified based on biological function using GeneTrail (right panel). B, top 10 proteins whose binding to 14-3-3 was increased most after dsRNA stimulation. Access no. = Swiss-Prot access number; FC = fold change in 14-3-3 binding relative to control. “Published” column shows whether 14-3-3 interaction had been published previously. C, HaCaT keratinocytes were transfected with pI:C for the indicated times, and the expression of RAI was analyzed from cell lysates via immunoblotting. The size of the intact RAI protein is approximately 90 kDa. D, the cells were transfected with control siRNA and RAI (gene name PPP1R13L) specific siRNA molecules for 24 h before stimulation with dsRNA for 5 h, after which the expression of RAI was detected via immunoblotting. The effect on RAI silencing on dsRNA-induced caspase-3 activation, tBid, and IκBα expression was detected. GAPDH detection was used to confirm the equal loading. E, the effect of RAI silencing on dsRNA-induced TNF cytokine expression was detected using quantitative RT-PCR. Comparable data were obtained from two separate experiments. *p < 0.05 versus pI:C-transfected ctr-siRNA-sample.