Figure 5

p-hnRNP I is localized to the cytoplasm and interacts with RoR at the hnRNP I binding motifs. (A) Subcellular localization of hnRNP I by cell fractionation. After cell fractionation of MCF-7 cells, an equal amount of protein (30 μg/lane) was loaded for each lane. Note that a very weak hnRNP I band was detected in the cytosolic fraction whereas a strong band came from the nuclear fraction. Topoisomerase I (topo I) serves as a nuclear protein marker; GAPDH as a cytosolic protein marker. (B) Detection of hnRNP I protein after RNA precipitation by western blot. The same cytosolic and nuclear extracts from (A) were used for RNA precipitation. However, since over 95% of hnRNP I is present in the nucleus, we adjusted the amount of protein and used the nuclear protein of about 1/5 amount of the cytosolic protein for RNA precipitation. (C) Detection of hnRNP I and p-hnRNP I by confocal immunofluorescence microscopy. While hnRNP I (red) is predominantly in the nucleus, p-hnRNP I (green) is in the cytoplasm. (D) PKA enhances hnRNP I phosphorylation. (E) RoR-bound hnRNP I is phosphorylated and PKA enhances the interaction of RoR with hnRNP I. Total cellular extract from the same transfected cells in (D) was used for RNA precipitation. The same membrane was probed simultaneously with hnRNP I antibody (rabbit origin) and p-hnRNP I antibody (mouse origin), followed by secondary antibody probing as indicated. (F) Serine phosphorylation of hnRNP I is essential for its interaction with RoR. MCF-7 cells were transfected with wild-type hnRNP I or mutant hnRNP I (S16A). Total cellular extract was prepared for RNA precipitation. (G) Suppression of p53 by RoR-oligo-1. MCF-7 cells were transfected with vector or E4-d4; or control oligo, oligo-1 or oligo-M. The transfected cells were then treated with doxo at 1 μg/ml for 24 h before protein extraction for western blot. (H) Precipitation of hnRNP I by biotin-labeled RNA oligos (Supplementary information, Figure S9B). Total cellular extract from non-transfected MCF-7 cells was prepared for RNA precipitation. The same membrane was probed simultaneously with hnRNP I antibody (rabbit origin) and p-hnRNP I antibody (mouse origin), followed by secondary antibody probing as indicated.