Plasmids encoding GFP-Nup98 or GFP alone were transfected into HEK293T cells. These proteins were immunoprecipitated from whole cell lysates using an antibody directed against GFP. …
(A) Curated protein-protein interactions (PPI) among identified Nup98 binding partners are represented in a PPI network. Edge thickness indicates the confidence score for the interaction and node …
Endogenous DHX9, hnRNP U, and Nup98 were immunoprecipitated from whole cell lysates (Input) using specific antibodies as indicated above the panels. Anti-GFP antibodies were used to assess …
The cellular distribution of Nup98, DHX9, and hnRNP U in HEK293T cells was examined by indirect immunofluorescence using antibodies directed against each protein as indicated. The positions of …
HEK293T cells were transfected with shRNA targeting Nup98 or with a control shRNA. Four days later the cellular distributions of Nup98 and either DHX9 or hnRNP U were examined by indirect …
HEK293T cells were transfected with a control shRNA or an shRNA targeting DHX9. Four days later the cellular distribution of Nup98 and DHX9 were examined by indirect immunofluorescence. Merged …
Four days after shRNA transfection, lysates from cells depleted of the indicated protein (listed above top panel) were analyzed by western blotting using antibodies directed against Nup98, DHX9, and …
(A) HEK293T cells expressing GFP-NUP98 were used to compare DHX9 and GFP-Nup98 localization by immunofluorescence microscopy. Two magnifications are shown, each showing that upon GFP-NUP98 …
HEK293T cells expressing GFP (A) or GFP-NUP98 (B) were used to compare their localization to DHX9 or hnRNP U detected by indirect immunofluorescence microscopy using specific antibodies. Merged …
Western blots of proteins derived from HEK293T cells lysates expressing GFP or GFP-Nup98 are shown. Antibodies were used for immunoblotting to detect the proteins indicated to the right of the …
(A) HEK293T cells expressing GFP-NUP981–497 were used to assay the localization of DHX9 detected by immunofluorescence microscopy. Two magnifications are shown and examples of GFP-Nup981–497 …
(A) Nup98 was affinity purified from HEK293T cell lysates. Bead-bound protein complexes were then incubated with or without RNase A, and proteins remaining bound to Nup98 were analyzed by western …
(A) Anti-DHX9 antibodies coupled to beads were used to immobilize recombinant DHX9. Bead-bound DHX9 was then incubated with recombinant Nup98 or GST in the presence or absence of RNA (poly I:C), …
The ATPase activity (ATP hydrolysis rate) of purified recombinant GST-DHX9 in the presence of RNA alone (no addition) or following the addition of increasing concentrations of GST-Nup98 or GST (show …
(A) The ATP hydrolysis rate (ATP/sec) of purified recombinant GST-DHX9 or GST alone in the presence or absence of RNA was examined. Error bars indicate standard deviation. Results from three …
Following crosslinking of HEK293T cells to preserve protein/RNA complexes, cell lysates were incubated with beads coupled to a control IgG (α-GFP) or beads coupled to Nup98 or DHX9 specific …
Western blot analysis of protein immunoprecipitation (IP) fractions described in Figure 8 to detect associated RNA. The indicated IP samples (IP IgG) were analyzed by western blotting using …
HEK293T cells were transfected with a control shRNA or an shRNA targeting Nup98 or DHX9. RNA immunopurified with Nup98 or DHX9 was reverse transcribed and used in qPCR reactions to assess the levels …
Western blot analysis of cell lysates and protein immunoprecipitation (IP) fractions described in Figure 9 to detect associated RNA. The indicated cell lysates and IP samples (list above the panels) …
(A) HEK293T cells were transduced with a control shRNA or an shRNA targeting Nup98 or DHX9. Cells were fractionated into nuclear and cytoplasmic samples and the levels of the indicated gene …
HEK293T cells stably expressing Dam-GFP, Dam-Nup98 or Dam-DHX9 were transduced with lentivirus encoding a control shRNA (white) or an shRNA targeting Nup98 or DHX9 (gray) and DamID analysis was …
HEK293T cells stably expressing Dam-GFP or Dam-Nup981–504 were transduced with lentivirus encoding a control shRNA (white) or an shRNA targeting DHX9 (gray), and Dam-ID analysis was performed. The …
HEK293T cells were transduced with a control shRNA or an shRNA targeting Nup98 or DHX9. RNA was purified and transcript levels from the indicated genes (x-axis) were reverse transcribed and …
HEK293T cells transfected with the luciferase gene under control of a cAMP-regulatory element (CRE) were co-transfected with two plasmids, one containing GFP-NUP98 or GFP and another containing …
Western blots of proteins derived from HEK293T cell lysates expressing the indicated DHX9 constructs were performed. These constructs contain a C-terminal HA-tag, allowing detection and comparison …
HEK293T cells were transfected with a control shRNA or an shRNA targeting Nup98 or DHX9. RNA from these cells was reverse transcribed and cDNAs used as template in qPCR reactions containing primers …
Supplemental information.
(A) Complete mass spectrometry data exported from PEAKS software. (B) List of plasmids. (C) List of shRNAs. (D) List of primers.