Figure 5.

SRP proteins interact with DDX3 and activate AREG mRNA translation

(A) In vivo translation assays of the AREG 5’+3′ reporter in SAS cells that were transfected with the indicated shRNAs. Bar graphs show the hFL/hRL activity (mean ± SD) of shRNA transfectants relative to control cells. ∗p < 0.01.

(B) The experiment was performed as in (A), except that AREG 3′ reporter and shRNAs targeting DDX3 or SRP components were used (mean ± SD; ∗p<0.01).

(C) SAS cells were transfected with shRNA as in (B). Immunoblotting was performed using antibodies as indicated.

(D) Immunoprecipitation of SAS cell lysates using control IgG or anti-DDX3 in the absence or presence of RNase, followed by immunoblotting.

(E and F) Immunofluorescence co-staining for DDX3 with GRP94 (E) or SRP54 (F) in SAS cells. The framed region in the upper images is magnified in the lower images. The histograms of fluorescence intensity across the pink lines are shown to the right. Scale bars, 20 μm.

(G) The lysates of siRNA-transfected SAS cells were subjected to immunoblotting using antibodies as indicated.