Fig 1: ATAD5 interacts with DEAD-box RNA helicases and DHX9. (A) HEK293T cells were transfected with strep-Tag II-ATAD5 cDNA. After 48 h, proteins were extracted and subjected to affinity purification-mass spectrometry analysis. The numbers of peptide hits for some selected proteins from the analysis were displayed. Proteins in red indicate DEAD-box RNA helicases and DHX9. (B–E, K) After transfection of HEK293T cells as indicated, nuclear extracts were prepared for immunoprecipitation (IP) with an anti-FLAG antibody. Immunoprecipitates were eluted, resolved by SDS-PAGE, and immunoblotted using the indicated antibodies. (F–I) Nuclear extracts from HEK293T cells were immunoprecipitated with anti-ATAD5 antibody (F, G) or anti-DDX5 antibody (H, I). (H, I) Cells were transfected with UAF1. (G, I) Relative band intensities were quantified and plotted, N=4. Statistical analysis: two-tailed paired Student's t-test; **** P < 0.001, **P < 0.01, *P < 0.05, ns, not significant. (J) Purified MYC-FLAG-DDX5, His-UAF1, and His-FLAG-ATAD5-N400 (ATAD5 1–400 aa) proteins were incubated as indicated and pulled down with an anti-MYC antibody. Purified FLAG-His-MBP-TALE protein was included as a negative control. (K) HEK293T cells were transfected with FLAG-tagged DDX5 and one of S-tagged UAF1 deletion mutants (S-UAF1WD40 and S-UAF1SLD). (L) HeLa-TetOn-RNaseH1-GFP cells were transfected with UAF1 siRNA, treated with doxycycline to express RNaseH1 for 48 h and immunostained with the S9.6 antibody. The intensity of S9.6 staining was quantified and plotted. Three independent experiments were performed, and one representative result was displayed. Red bar indicates mean value. Statistical analysis: two-tailed Student's t-test; * P < 0.05 and ns, not significant.
Fig 2: ATAD5 depletion reduces the association of RNA helicases with normal and stalled replication forks. (A) HEK293T cells were labeled with EdU for 20 min and then treated with 10 µM thymidine for indicated times. Whole cell extracts were prepared for an iPOND EdU-thymidine chase assay. (B) HEK293T cells were transfected with ATAD5 siRNA for 48 h then whole cell extracts were prepared for an iPOND assay. (C–F) U2OS-TetOn-ATAD5 cell lines were transfected with ATAD5 siRNAs and treated with doxycycline to express wild type ATAD5 (ATAD5WT) or UAF1 interaction defective mutant ATAD5 (ATAD5?UAF1). After 48 h, cells were fixed for a SIRF assay on DDX21 (C, D) or DDX5 (E, F). Hydroxyurea (HU) was added at a final concentration of 2 mM for 3 h before fixation. (C, E) Representative SIRF images. (D, F) Number of nuclear SIRF signal foci were counted and plotted. Three independent experiments were performed and one representative result is displayed. Red bar indicates mean value. Statistical analysis: two-tailed Student's t-test; **** P < 0.001, *** P < 0.005, **, P < 0.01, * P < 0.05 and ns, not significant.
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