Journal: Cancer cell
Article Title: Elevated CXorf67 Expression in PFA Ependymomas Suppresses DNA Repair and Sensitizes to PARP Inhibitors
Figure Lengend Snippet: CXorf67 Interacts with the PALB2 WD40 Domain through the PALB2-Binding Motif (A) Immunoblot detection of anti-CXorf67 immunoprecipitates. Daoy CXorf67 WT and KO cells were treated with CPT (0.1 μM) for 1 h and lysates treated with Benzonase were precipitated with anti-CXorf67 and analyzed by immunoblotting with the indicated antibodies. (B) Direct binding between recombinant GST-WD40 and His-CXorf67 in vitro . GST-agarose beads-bound GST-WD40 or GST proteins were incubated with purified His-CXorf67. After wash, the beads-bound proteins were subjected to western blot analysis using anti-GST and anti-His antibodies. (C) Sequence alignment showing the similarity between CXorf67 and BRCA2 in a minimal PALB2-binging motif (top). Pull-down (PD) assays were performed by mixing purified recombinant GST-WD40 protein and biotinylated WT C67 (420–432) peptide or W425C mutant peptide for 2 h. The peptides were pulled down using streptavidin agarose beads followed by immunoblotting analyses. (D) PALB2 co-immunoprecipitation with CXorf67 is lost in CXorf67 W425C mutant. Flag-tagged CXorf67 WT or W425C mutant were co-expressed with Myc-tagged PALB2 in HEK293T cells. CXorf67 was immunoprecipitated with an anti-Flag antibody. (E) U2OS cells expressing EGFP-PALB2, EGFP-C67-WT, or W425C mutant were laser micro-irradiated and monitored using a live-cell imaging microscope. Representative confocal images are shown. White arrows indicate irradiated regions. Accumulation of EGFP-PALB2, EGFP-C67-WT, and W425C mutant on the DNA damage tracks was quantified. Data represent the mean values (±SD) from three independent experiments. (F) CXorf67 W425C cannot inhibit Rad51 foci formation. Daoy C67-KO cells were transfected with HA-tagged WT CXorf67 or the W425C mutant. The cells were then treated with CPT (0.1 μM) for 4 h and stained with anti-Rad51 antibody followed by Cy3-conjugated secondary antibody. Scatter dot plot represents the Rad51 foci per nuclei. Data are presented as mean ± SEM (n = 104–113; unpaired t test; ns, not significant; *p
Article Snippet: Excess of peptides were incubated with 30 μl of Streptavidin Agarose beads (SA100–04, Invitrogen) pre-equilibrated with binding buffer (20 mM HEPES, pH 7.5, 100 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.01% IGEPAL CA-630) for 1 h at 4°C.
Techniques: Binding Assay, Recombinant, In Vitro, Incubation, Purification, Western Blot, Sequencing, Mutagenesis, Immunoprecipitation, Expressing, Irradiation, Live Cell Imaging, Microscopy, Transfection, Staining