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. 2013 Sep 4;8(9):e73752. doi: 10.1371/journal.pone.0073752

Figure 4. DDX1 interferes with KSRP association with 14-3-3 and competes with 14-3-3 for interaction with KSRP.

Figure 4

(A) HeLa-TO cells were cotransfected with a FLAG-KSRP expression vector or a control FLAG vector and a control siRNA or a DDX1 siRNA. Cytoplasmic extracts were subjected to anti-FLAG immunoprecipitation. The precipitates were analyzed by anti-14-3-3 and anti-FLAG immunoblotting. 5% input was also analyzed by anti-14-3-3. (B) HeLa-TO cells were cotransfected with a FLAG-KSRP expression vector or a control FLAG vector and DDX1 siRNA. Cytoplasmic extracts were subjected to anti-FLAG immunoprecipitation with the addition of two different concentrations (6 and 12 nM) of recombinant GST-DDX1 or GST. The precipitates were analyzed by anti-14-3-3 and anti-FLAG immunoblotting. (C) HeLa-TO cells were transfected with vectors expressing FLAG-KSRP or FLAG-KSRP fragments consisting of the four KH motifs or the C-terminus. The FLAG immunoprecipitations were subjected to immunoblotting with anti-14-3-3, anti-DDX1, and anti-FLAG. (D) HeLa-TO cells were transfected with a control FLAG vector or a FLAG-DDX1 expression vector. Cytoplasmic extracts were subjected to anti-FLAG immunoprecipitation. The precipitates were analyzed by anti-14-3-3 and anti-FLAG immunoblotting. 5% input was also analyzed by anti-14-3-3.