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

Figure 3. Subcellular localization of KSRP is regulated by DDX1.

Figure 3

(A) HeLa-TO cells were transfected with a control siRNA or a DDX1 siRNA. Cytoplasmic and nuclear extracts from equal number of cells were subjected to immunoblot analysis with anti-KSRP, anti-DDX1, anti-HuR, anti-AUF1, or anti-14-3-3 which recognizes all isoforms. Antibodies against cytoplasmic α-tubulin and a nuclear protein, origin recognition complex subunit 2 (ORC2), were also used as controls for subcellular fractionation. Two independent transfection experiments were carried out. Quantification of KSRP levels in the cytoplasmic and nuclear fractions is indicated. (B) The nuclear extracts used in (A) were diluted 5-fold and subjected to immunoblot analysis with anti-KSRP and anti-ORC2. Quantification of the nuclear KSRP levels is indicated. (C) Total extracts of cells transfected with CAT or DDX1 siRNAs were analyzed by anti-KSRP or anti-HuR (D to F). HeLa-TO cells were transfected with CAT siRNA or DDX1 siRNA (D), CAT siRNA or DDX1 siRNA and a construct expressing FLAG-KSRP (E), or CAT siRNA or DDX1 siRNA and a construct expressing EGFP-KSRP (F). Transfected cells were analyzed by anti-KSRP (D), anti-FLAG (E), or GFP signal (F) (top two rows), and by DAPI staining (bottom two rows).