Fig 1: PR100 interacts with DEAD-box RNA helicases in an RNA-dependent manner.a, b Purified recombinant GST (−) or GST-FLAG-PR100 (+)-bound glutathione beads were mixed with (+) or without (−) NSC-34 cell lysates. After 18 h of rotation at 4 °C, the glutathione beads were washed, fractionated by 5-20% gradient-gel SDS-PAGE, and stained with Coomassie brilliant blue (CBB) (a). PR100-binding proteins (bands 1–15) were isolated and identified by mass-spectrometry analysis. Identified proteins were categorized by PANTHER Classification System (b). c NSC-34 cell lysates overexpressing HA-tagged DDX5, DDX17, DDX18, or DDX21 and purified recombinant GST or GST-FLAG-PR100 (GF-PR)-bound glutathione beads were incubated with or without 20 μg/mL RNase A. After the incubation, the cell lysates were mixed with recombinant GST or GST-FLAG-PR100-bound glutathione beads. The glutathione beads were washed and subjected to immunoblotting (IB) using indicated antibodies. The large smear within the molecular weights ranging 25–46 kDa is thought to consist of C-terminal truncated GST-FLAG-PR100 proteins. A band located around 50 kDa in the GST lane is thought to be dimerized GST and/or aggregated GST-derived proteins. PD, pull down d Purified recombinant GST (−) or GST-FLAG-PR100 (+)-bound glutathione beads were mixed with (+) or without (−) NSC-34 cell lysates. After 4 h of rotation at 4 °C, the glutathione beads were washed and subjected to immunoblotting (IB) using indicated antibodies. The large smear within the molecular weights ranging 25–46 kDa is thought to consist of C-terminal truncated GST-FLAG-PR100 proteins. A band located around 50 kDa in the GST lane is thought to be dimerized GST and/or aggregated GST-derived proteins. e, f NSC-34 cells, transiently transfected with indicated vectors, were harvested at 48 h after the transfection and the prepared cell lysates were subjected to immunoprecipitation (IP) with the FLAG, control (e), or HA (f) antibody. The washed precipitates were fractionated by SDS-PAGE, followed by immunoblotting (IB). The precipitates were also spotted onto PVDF membranes for dot blotting analysis. g NSC-34 cells overexpressing EGFP-FLAG-PR100 (green) together with HA-tagged DDX5, DDX17, DDX18, or DDX21 were fixed and immunostained with HA (red). Nuclei were stained with Hoechst33258 or DAPI (blue). Scale bar: 20 μm